GENETICALLY ENCODED SYSTEM FOR CONSTRUCTING AND DETECTING BIOLOGICALLY ACTIVE AGENTS

Abstract

This invention relates to the field of genetic engineering. Specifically, the invention relates to the construction of operons to produce biologically active agents. For example, operons may be constructed to produce agents that control the function of biochemical pathway proteins (e.g., protein phosphatases, kinases and/or proteases). Such agents may include inhibitors and modulators that may be used in studying or controlling phosphatase function associated with abnormalities in a phosphatase pathway or expression level. Fusion proteins, such as light activated protein phosphatases, may be genetically encoded and expressed as photoswitchable phosphatases. Systems are provided for use in controlling phosphatase function within living cells or in identifying small molecule inhibitors/activator/modulator molecules of protein phosphatases associated with cell signaling.

Claims

1. An engineered bacterial cell, comprising: a bacterial two-hybrid system for linking expression of a reporter protein to production of an inhibitor of a protein phosphatase by the engineered bacterial cell; and an isoprenoid pathway for producing the inhibitor of the protein phosphatase.

2. The engineered bacterial cell of claim 1, wherein the bacterial two-hybrid system comprises one or more nucleic acid sequences constitutively expressing a first fusion protein and a second fusion protein, wherein the first fusion protein is configured to associate with the second fusion protein in situ to form a complex when the engineered bacterial cell has produced the inhibitor of the protein phosphatase in a sufficient quantity to elicit a biochemical change in the first fusion protein such that formation of the protein complex is possible.

3. The engineered bacterial cell of claim 2, wherein the one or more nucleic acid sequences comprises an operon, a promoter, and a reporter gene, wherein the promoter is configured to drive expression of a reporter protein from the reporter gene when the operator is bound by the complex, thereby linking the expression of the reporter protein with the production of the inhibitor of the protein phosphatase by the engineered bacterial cell.

4. The engineered bacterial cell of claim 1, wherein the bacterial two-hybrid system comprises one or more components that encode: a substrate recognition domain coupled to a DNA-binding protein; a substrate domain coupled to a subunit of RNA polymerase; a protein kinase; a protein phosphatase; and an operon comprising: an operator for the DNA-binding protein; a binding site for the RNA polymerase; and a reporter gene encoding the reporter protein.

5. The engineered bacterial cell of claim 4, wherein the substrate recognition domain comprises a substrate homology 2 (SH2) domain.

6. The engineered bacterial cell of claim 4, wherein the DNA-binding protein comprises a 434 phage cI repressor.

7. The engineered bacterial cell of claim 4, wherein the substrate domain comprises a peptide substrate of the protein kinase and the protein phosphatase.

8. The engineered bacterial cell of claim 4, wherein the subunit of the RNA polymerase comprises an omega subunit of RNA polymerase (RP?).

9. The engineered bacterial cell of claim 4, wherein the protein kinase comprises a Src kinase.

10. The engineered bacterial cell of claim 4, wherein the one or more components of the bacterial two-hybrid system further encodes a molecular chaperone.

11. The engineered bacterial cell of claim 10, wherein the molecular chaperone comprises an Hsp90 co-chaperone from H. sapiens or a CDC37.

12. The engineered bacterial cell of claim 4, wherein the operator comprises a 434 phage cI operator.

13. The engineered bacterial cell of claim 4, wherein the reporter protein confers antibiotic resistance.

14. The engineered bacterial cell of claim 13, wherein the reporter protein conferring antibiotic resistance comprises an enzyme that allows the engineered bacterial cell to inactivate an antibiotic.

15. The engineered bacterial cell of claim 4, wherein the protein phosphatase comprises protein tyrosine phosphatase 1B.

16. The engineered bacterial cell of claim 1, further comprising a synthase enzyme for synthesizing the inhibitor of the protein phosphatase.

17. The engineered bacterial cell of claim 16, wherein the synthase enzyme is a terpene synthase enzyme.

18. The engineered bacterial cell of claim 16, wherein the synthase enzyme is wild-type.

19. The engineered bacterial cell of claim 16, wherein the synthase enzyme has at least one mutation relative to a wild-type synthase enzyme.

20. The engineered bacterial cell of claim 1, wherein the inhibitor comprises a terpenoid compound or a structural variant of a terpenoid compound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0037] FIG. 1A-G illustrates embodiments and shows exemplary results of developing a photoswitchable phosphatase, e.g. PTP1B.sub.PS.

[0038] FIG. 1A illustrates one embodiment of a design of PTP1B.sub.PS: Light-induced unwinding of the A? helix of LOV2 destabilizes the ?7 helix of PTP1B and, thus, inhibits catalysis. FIG. 1B illustrates one embodiment of Elaboration: In the competitively inhibited structure of PTP1B (orange), the ?7 helix is stable, and the WPD loop (black) adopts a closed, catalytically competent conformation. In the apo structure (yellow), the ?7 helix is disordered, and the WPD loop (blue) adopts an open, inactive conformation. We attached the C-terminal ?7 helix of PTP1B (SEQ ID NO: 1) to the N-terminal A? (SEQ ID NO: 2) helix of LOV2 at homologous crossover points (1-7) to create a chimera for which the photoresponsive of LOV2 destabilizes the ?7 helix. FIG. 1C shows exemplary results of optimization of one embodiment: Construct 7 exhibited the largest dynamic range of the crossover variants; 7.1 had an improved activity over 7, while 7.1(T406A) had an improved dynamic range over 7.1. FIG. 1D shows an exemplary analysis of the activity of PTP1B.sub.PS on pNPP indicates that light affects k.sub.cal, but not K.sub.m. FIG. 1E shows the dynamic range of PTP1B.sub.PS is similar for substrates of different sizes. FIG. 1F shows exemplary illustrations of two small molecules: p-nitrophenyl-phosphate (or pNPP), and 4-methylumbelliferyl phosphate (or 4MU) and a peptide domain from EGFR. FIG. 1G shows exemplary activity of PTP1B-LOV2 chimeras that differ in (A-D) crossover location and (E-E4) linker composition in the presence and absence of 455 nm light. Substrate: 4-methylumbelliferyl phosphate.

[0039] FIG. 2A-J shows exemplary biophysical characterizations of PTP1B.sub.PS.

[0040] FIG. 2A shows exemplary mutations that (i) prevent the formation of the cysteine adduct in LOV2 (C450M), (ii) destabilize the A? and J? helices if LOV2 (I532E, 1539E, and ?J?), or (iii) disrupt the allosteric network of PTP1B (Y152A/Y153A) reduced the photosensitivity of 7.1 and, with the exception of 1532E and C450M, lowered its specific activity. FIG. 2B shows exemplary exposure of PTP1B.sub.PS to 455 nm light reduces its ?-helical content (CD.sub.222 nm). FIG. 2C shows exemplary optical modulation of ?-helical content (i.e., ?.sub.222=CD.sub.222-dark?CD.sub.222-light) is necessary, but not sufficient for optical modulation of catalytic activity. The dashed line denotes ?.sub.222 for an equimolar solution of PTP1B.sub.WT and LOV2.sub.WT. FIG. 2D shows exemplary fluorescence of six tryptophan residues in the catalytic domain of PTP1B which enables optical monitoring of its conformational state. FIG. 2E-F shows exemplary thermal recovery of (FIG. 2E) ?-helical content and (FIG. 2F) tryptophan fluorescence of PTP1B.sub.PS. FIG. 2G shows exemplary kinetic constants for thermal resetting are larger for ?-helical content than for tryptophan fluorescence, suggesting that LOV2 resets more quickly than the PTP1B domain. This discrepancy is smallest for the most photosensitive variant: 7.1(T406A). FIG. 2H shows exemplary alignments of the crystal structures of PTP1B.sub.PS (blue) and apo PTP1B.sub.WT (orange) indicate that LOV2 does not distort the structure of the catalytic domain. The LOV2 domain of PTP1B.sub.PS could not be resolved; a flexible loop at the beginning of the ?7 helix likely causes LOV2 to adopt variable orientations in the crystal lattice. The ?6 and ?7 helices of an inhibited structure of PTP1B (yellow) are shown for reference. FIG. 21 shows an exemplary gap in the crystal structure of PTP1B.sub.PS that can accommodate LOV2. FIG. 2J shows where exemplary crystals of a PTP1B-LOV2 fusion are green and turn clear when illuminated with 455 nm light; LOV2 is, thus, unequivocally present. Error bars for A, C, and G denote standard error (n>3). Note: PTP1B.sub.PS corresponds to construct 7.1(T406A) from FIG. 1.

[0041] FIG. 3A-D demonstrates exemplary Fluorescence-based Biosensors having PTP1B activity.

[0042] FIG. 3A shows one embodiment of a sensor for PTP1B activity. This sensor consists of a kinase substrate domain, a short flexible linker, and a phosphorylation recognition domain, sandwiched between two fluorescent proteins (e.g., a cyan fluorescent protein and a yellow fluorescent protein). When the sensor is in its unphosphorylated state, Forster resonance energy transfer (FRET) between the two fluorophores causes a decrease in CFP fluorescence and an increase in YFP fluorescence; when the sensor is in its phosphorylated state, the absence of FRET causes the opposite effect. FIG. 3B shows an exemplary increase in the ratio of donor fluorescence (CFP) to acceptor fluorescence (YPet) evidences the presence of Src kinase (i.e., a tyrosine kinase). When either (i) EDTA, which chelates a metal cofactor of Src, or (ii) PTP1B, which dephosphorylates the substrate domain, are additionally added, this increase does not occur. FIG. 3C shows one embodiment as another variety of the FRET sensor for A; this one uses mClover3 and mRuby3. The excitation and emission wavelengths of these proteins make them compatible with LOV2-based imaging experiments. FIG. 3D shows an exemplary repeat of the experiment from B with the sensor from C.

[0043] FIG. 4A-H demonstrates exemplary Evidence of phosphatase activity within living cells using photoconstructs and fluorescent tags.

[0044] FIG. 4A-C shows embodiments of three constructs are expressed in Cos-7 cells: (FIG. 4A) GFP-PTP1B.sub.PS, (FIG. 4B) GFP-PTP1B.sub.PS-A, and (FIG. 4C) GFP-PTP1B.sub.PS-B. Here, GFP-PTP1B.sub.PS is a fusion of green fluorescent protein (GFP) and the N-terminus of 7.1(T406A) from FIG. 1B-C(without the histidine tag); GFP-PTP1B.sub.PS-A is a fusion of GFP-PTP1B.sub.PS and the C-terminal domain of full-length PTP1B; and GFP-PTP1B.sub.PS-B is fusion of GFP-PTP1B.sub.PS and the C-terminal endoplasmic reticulum (ER) anchor of full-length PTP1B (see below). GFP-PTP1B.sub.PS localizes to the cytosol and nucleus, while GFP-PTP1B.sub.PS-A and GFP-PTP1B.sub.PS-B localize to the ER. FIG. 4D-H shows exemplary results of cell-based studies of PTP1B.sub.PS. We transformed Cos-7 cells with a plasmid containing (i) the FRET sensor from FIGS. 3C-3D and (ii) PTP1B.sub.PS or PTP1B.sub.PS/C450M (a light-insensitive mutant). In this experiment, we illuminated individual cells with 447 nm light and immediately imaged them with 561 nm light. Light-modulated changes in FRET ratio (as defined in FIG. 3) allowed us to detect light-modulated changes in PTP1B activity. FIG. 4D-E shows an exemplary Cos-7 cell transformed with PTP1B.sub.PS at two time points: (FIG. 4D) immediately after excitation with 447 nm light and (FIG. 4E) after 1 min. A slight increase in FRET ratio (dark green to lighter green) evidences photoactivation of PTP1B. (F-G). A Cos-7 cell transformed with PTP1B.sub.PS (C450M) at two time points: (FIG. 4F) immediately after excitation with 447 nm light and (G) after 1 min. The absence of a detectable change in FRET-ratio indicates that the change observed in D-E results from light-induced changes in PTP1B activity. FIG. 4H shows an exemplary average fractional change in FRET ratio observed in the nucleus (nuc) and cytosol (cyt) after 1 min and 2.67 min. The change is higher for PTP1B.sub.PS than for PTP1B.sub.PS(C450M), the light-insensitive mutant. Error bars indicate standard error.

[0045] FIG. 5A-C illustrates embodiments of drug discovery.

[0046] FIG. 5A shows an exemplary use of a phosphatase, i.e. drug target (upper left depiction of PTP1B) for identifying a synthetic enzyme (lower right depiction) where the enzyme is then used for providing an inhibitor or modulatory molecule for the phosphatase, thus showing a general framework for using enzymes to build inhibitors of chosen protein targets. FIG. 5B shows an exemplary analysis of structural relationships between binding pockets. A matrix compares individual properties (e.g., volume) between binding pocket 1 and all other binding pockets (2 to n) capable of functionalizing (e.g., P450) or binding to (e.g., PTP1B) ligands synthesized within pocket 1. FIG. 5C shows an exemplary comparison of the ability of binding pockets in a biosynthetic pathway to bind to intermediates.

[0047] FIG. 6 illustrates PTP1B showing an overlay of allosterically inhibited (green) and competitively inhibited (orange) structures of PTP1B (PDB entries 1t4j and 2f71, respectively) show activity-modulating conformational changes: Unwinding of the ?7 helix of LOV2 (blue) causes its catalytically essential WPD loop (right) to adopt an open, catalytically compromised conformation. Competitive (red) and allosteric (yellow) inhibitors highlight the active site and allosteric site, respectively.

[0048] FIG. 7A-B shows and exemplary analysis of binding affinity. FIG. 7A shows embodiments of two binding partners of PTP1B: LMO4 and Stat3. FIG. 7B shows an exemplary binding isotherm based on binding-induced changes in the tryptophan fluorescence of PTP1B (the ligand isTCS 401, a competitive inhibitor).

[0049] FIG. 8A-B illustrates exemplary structural alignment of PTP1B (light blue) and STEP (orange), FIG. 8A, which have only 31% sequence identity, shows remarkable structural similarity. FIG. 8B illustrates an exemplary structure of PTK6. Both STEP and PTK6 possess a C-terminal alpha-helix that is compatible with actuation by the N-terminal helix of LOV2 (i.e., an photomodulatory architecture similar to that depicted in FIG. 1).

[0050] FIG. 9A-B illustrates an exemplary framework for building an enzyme modulated by red light. We will attach the C-terminal ?-helix of PTP1B to the N-terminal ?-helix of BphP1.

[0051] FIG. 10A-B illustrates an exemplary operon for screening photoswitchable variants of PTP1B. FIG. 10A shows an exemplary illustration where in its active state (here the far-red state), PTP1B dephosphorylates the substrate domain, prevents substrate-SH2 association, and, thus, prevents transcription. FIG. 10B shows an exemplary illustration where in its inactive state (here, the red state), the phosphorylated substrate domain binds SH2, permitting transcription of a gene for antibiotic resistance.

[0052] FIG. 11A-B illustrates an exemplary strategy for evolution of photoswitchable proteins. FIG. 11A illustrates where we will compare the growth of colonies on replicate plates exposed to red and infrared light and select colonies that exhibit differential growth. FIG. 11B illustrates where we will further characterize the photosensitivity of top hits in liquid culture.

[0053] FIG. 12A-B illustrates an exemplary FRET-based sensor developed for measuring intracellular phosphatase or kinase activity. Binding of the substrate and SH2 domain either (FIG. 12A) enhance or (FIG. 12B) reduce FRET, depending on architecture.

[0054] FIG. 13 shows a cartoon of imaging experiments. We will inactivate PTP1B PS within subcellular regions (1-10 m) containing different amounts of plasma membrane, ER, and cytosol, and we will use fluorescence lifetime imaging to examine the phosphorylation state of our FRET-based sensor (from FIG. 12) throughout the cell.

[0055] FIG. 14A-D illustrates an exemplary starting point for lead drug design and discovery.

[0056] FIG. 14A illustrates Abietic acid. FIG. 14B demonstrates inhibition of PTP1B by abietic acid at concentrations (dark to light) of 0-400 uM. Analysis of different fits suggests noncompetitive or mixed-type inhibition. FIG. 14C illustrates Abietic acid (green) docked in the allosteric site of PTP1B. We have since shown that abietic acid binds to the active site of PTP1B. Inset highlights active site in black. FIG. 14D shows an exemplary X-ray crystal structure of a known allosteric inhibitor (blue).

[0057] FIG. 15A-D illustrates an exemplary FIG. 15A Pathway for the synthesis of terpenoids (mevalonate can be synthesized through pMevT or added to the media). FIG. 15B shows exemplary Abietadiene titers generated by E. Coli DH5a transformed with the plasmids from A (with no P450). FIG. 15C-D shows exemplary GC-MS analysis of products of FIG. 15C) abietadiene-producing strain and FIG. 15D abietic-acid-producing strain: (1) abietadiene, (2) levopimaradiene, and (3) abietic acid (ion counts in 10,000 for C and 1,000 for FIG. 15D). Note: E. Coli DH5a avoids protein overexpression is commonly used in metabolic engineering*.sup.4.

[0058] FIG. 16A-B illustrates exemplary terpenoids showing differences in stereochemistry, shape, size, and chemical functionality. FIG. 16A illustrates clockwise from abietic acid (1), neoabietic acid (2), levopimaric acid (3), dihydroabietic acid (4). FIG. 16B shows exemplary initial rates in PTP1B on 10 mM of p-NP phosphate in the presence of 200 uM inhibitor. No inhibitor. Error bars=standard error (n>5).

[0059] FIG. 17A-C shows results from exemplary studies. FIG. 17A N-HSQC spectra of PTP1B (red) and PTP1B bound to abietic acid (blue). Inset: Crystal structure of PTP1B. FIG. 17B-C shows exemplary Tryptophan (W) fluorescence of PTP1B in the presence of FIG. 17B culture extract of control (ABSx), abietadiene-producing (ABS), and abietic-acid producing (ABS/BM3) strains and (FIG. 17C) various concentrations of abietic acid and 25 uM of known allosteric inhibitor (BBR). Error bars represent standard error (n>5).

[0060] FIG. 18A-B illustrates exemplary terpenoids that differ in FIG. 18A stereochemistry and FIG. 18B shape. Inset: residues targeted for mutagenesis in class I site of ABS.

[0061] FIG. 19A-E illustrates exemplary terpenoids FIG. 19A carboxylated, FIG. 19B hydroxylated, FIG. 19C and halogenated diterpenoids. FIG. 19D-E shows exemplary residues targeted for mutagenesis in FIG. 19D P450 BM3 and FIG. 19E SttH.

[0062] FIG. 20 illustrates an exemplary WaterMap analysis of UPPS. Colors of water molecules correspond to free energies, relative to bulk water.

[0063] FIG. 21A-E illustrates an exemplary high-throughput screens for PTP1B inhibitors.

[0064] FIG. 21A Growth-coupled (i.e., selection; strategy 1). FIG. 21B) FRET sensor for PTP1B activity (strategy 2). FIG. 21C) FRET sensor and FIG. 21D) tryptophan fluorescence for changes in PTP1B conformation (strategies 3 and 4). FIG. 21E. Results for an operon similar to that shown in FIG. 21A, where Amp is replaced with Lux. Error bars=SD (n?3).

[0065] FIG. 22A-D illustrates exemplary inhibition of PTP1B. Error bars in FIG. 22C denote SE (n?3 independent reactions).

[0066] FIG. 22A shows exemplary alignments of the backbone of PTP1B in competitively inhibited (yellow and orange, PDB entry 2F71) and allosterically inhibited (gray and black, PDB entry 1T4J) poses. The binding of substrates and competitive inhibitors to the active site causes the WPD loop to adopt a closed (orange) conformation that stabilizes the C-terminal alpha7 helix through an allosteric network; this helix is unresolvable in allosterically inhibited, noncompetitively inhibited, and uninhibited structures, which exhibit WPD-open conformations (black). FIG. 22B shows an exemplary illustration of a chemical structure of abietic acid (AA). FIG. 22C shows exemplary initial rates of PTP1B-catalyzed hydrolysis of pNPP in the presence of increasing concentrations of AA. Lines show a fit to a model for mixed inhibition. FIG. 22D an exemplary illustration of this model, where the inhibitor (I) binds to the enzyme (E) and enzyme-substrate complex (ES) with different affinities.

[0067] FIG. 23A-C illustrates an exemplary NMR analysis of PTP1B-AA association.

[0068] FIG. 23A shows exemplary weighted differences in chemical shifts (??) between 1 H-15 N-HSQC spectra collected in the absence and presence of AA (PTP1B:AA of 10:1). The dashed red line delineates the threshold for values of Ad larger than two standard deviations (a) above the mean; gray bars mark residues for which chemical shifts broadened beyond recognition. FIG. 23B illustrates an exemplary crystal structure of PTP1B (PDB entry 3A5J, gray) highlights the locations of assigned residues (blue); inhibitors in the allosteric site (PDB entry 1T4J, green) and active site (PDB entry 3EB1, yellow) are overlaid for reference. Residues with significant CSPs (i.e., ??>?? mean+2?) are distributed across the protein (red) and, with the exception of two residues in the WPD loop, outside of known binding sites. FIG. 23C illustrates an exemplary detail of the active site (upper panel) and known allosteric site (lower panel) with inhibitors from (FIG. 23B) overlaid.

[0069] FIG. 24A-C illustrates an exemplary mutational analysis of the AA binding site.

[0070] FIG. 24A illustrates an exemplary crystal structure of PTP1B (gray, PDB entry 3A5J) shows the location of mutations introduced at five sites: the active site (red), the allosteric site (green), site 1 (orange), site 2 (yellow), and the L11 loop (blue). The bound configurations of BBR (allosteric site, PDB entry 1T4J) and TCS401 (active site, PDB entry 1C83) are overlaid for reference. FIG. 24B illustrates exemplary disruptive mutations introduced at each site. Mutations were designed to alter the size and/or polarity of targeted residues. The mutation denoted YAYA (Y152A/Y153A), which was identified in a previous study, attenuates allosteric communication between the C-terminus and the WPD loop. FIG. 24C illustrates exemplary fractional change in inhibition (F in Eq. 1) caused by the mutations from (B). Five mutations distributed across the protein reduced inhibition by AA and TCS401, but had negligible effect on inhibition by BBR. The similar effects of most mutations on AA and TCS401 suggest that both inhibitors bind to the active site. Error bars denote SE (propagated from n?9 independent measurements of each V in Eq. 1).

[0071] FIG. 25A-D illustrates exemplary computational analysis of AA binding.

[0072] FIG. 25A-B illustrates exemplary results of molecular dynamics simulations: backbone traces of PTP1B in (A) AA-free and FIG. 25B illustrates exemplary amino acid (AA)-bound states. The thickness of traces indicates the amplitude and direction of local motions (Methods). The binding of AA increases the flexibility of the WPD, E, and L10 loops. The WPD and L10 loops contain residues with significant CSPs (red), suggesting consistency between the results of MD and NMR analyses. FIG. 25C illustrates an exemplary representative bound conformation if AA (green). Upon binding to the active site, AA (i) forms a hydrogen bond with R221 that weakens a bond between R221 and E115 and (ii) prevents the formation of a hydrogen bond (red) between W179 and R221 that forms when the WPD loop closes. Both effects enhance the conformational dynamics of the WPD loop. FIG. 25D shows exemplary results of docking calculations are consistent with mixed-type inhibition: the binding of AA prevents the WPD loop from closing and disrupts, but does not preclude, the binding of pNPP (blue spheres).#

[0073] FIG. 26A-C illustrates exemplary terpenoids showing differences in stereochemistry, shape, size, and chemical functionality. FIG. 26A) Structural analogues of abietic acid (AA): continentalic acid (CA), isopimaric acid (IA), dehydroabietic acid (DeAA), and dihydroabietic acid (DiAA). FIG. 26B) Differences in degree of saturation yield pronounced differences in potency (i.e., IC 50), but not selectivity. Error bars represent 95% confidence intervals. FIG. 26C shows binding of three of the analogues depicted in FIG. 26A.

[0074] FIG. 27A-C Analysis of pathologically relevant mutations.

[0075] FIG. 27A illustrates an exemplary Histogram of kinetically characterized mutations. All mutations proximal (<4 A) to five or more network residues were influential (i.e., they altered k.sub.cat or K.sub.M by >50% or had a detectable influence on inhibition); non-consequential mutations, by contrast, had fewer neighboring network residues. FIG. 27B illustrates an exemplary crystal structure of PTP IB (gray, PDB entry 3A5J) highlights the locations of influential mutations on network residues; colors indicate whether they were introduced in biophysical studies or found in diseases. FIG. 27C illustrates an exemplary two cumulative distribution functions describe numbers of network residues proximal to (i) mutations identified in diseases and (ii) a random selection of sites. The two distributions are indistinguishable from one another (P<0.05), suggesting that disease-associated mutations do not occur preferentially near the allosteric network.

[0076] FIG. 28A-D illustrates and shows exemplary data using a Genetic operon linking PTP activity to the output of a gene of interest (GOI).

[0077] FIG. 28A shows embodiments of Operon A. An example of the operon. S, tyrosine substrate; P, phosphate group; cI, the 434 phage cI repressor; RpoZ and RP.sub.?, the omega subunit of RNA polymerase; cI OP, the binding sequence for the 434 phage cI repressor; and RB, the binding site for RNA polymerase (RNAP). Phosphorylation of the tyrosine substrate (by c-Src kinase) causes binding of the substrate-RP.sub.? fusion to the SH2-cI fusion; this binding event, in turn, localizes the RNA polymerase to RB, triggering transcription of the GOI. PTP1B dephosphorylates the substrate domain, preventing the association of substrate-RP.sub.? fusion and the SH2-cI, thereby, halting transcription of the GOI. Inactivation of PTP1B, in turn, re-enables transcription of the GOI. FIG. 28B illustrates one embodiment of a proposed medium-throughput screen for membrane-permeable inhibitors: A strain of the E. coli is transformed with the operon and grown in the presence of small molecules; small-molecule inhibitors of PTP1B modulate transcription of the GOI (e.g., a gene for luminescence, fluorescence, or antibiotic resistance) in a dose-dependent manner. The bar graph shows a predicted trend in data. FIG. 28C shows embodiments of Operon B. An operon that enables screens for selective inhibitors. This operon comprises operon A with (i) a second substrate-protein fusion (red), a decoy, that can bind to the SH2-cI fusion but not specifically to DNA or RNA polymerase, and (ii) a second PTP (e.g., TC-PTP) that is active on the substrate domain of the decoy. Because complexes between the decoy and SH2-cI do not trigger transcription, the decoy inhibits transcription by competing with cI-substrate for binding sites. Accordingly, molecules that inhibit PTP1B, but not TC-PTP (which dephosphorylates the decoy)that is, selective inhibitors-cause the greatest transcriptional activation. Molecules that inhibit both enzymes, by contrast, cause less activation. FIG. 28D shows embodiments of Operon C. This operon enables screens for photoswitchable enzymes. This operon comprises a version of operon A in which PTP1B has been replaced with a photoswitchable version of PTP1B. In this case, transcription of the GOI is different (e.g., higher or lower) under different sources of light. In the example shown, light inhibits the activity of a PTP1B-LOV2 chimera and, thus, enhances transcription of the GOI.

[0078] FIG. 29A-B shows exemplary Preliminary results showing phosphorylation-dependent expression of a GOT. FIG. 29A shows one embodiment of Operon A in which the GOI is a bacterial luciferase (LuxAB). PTP1B inhibits luminescence (i.e., reduces transcription of the GOI), while a catalytically inactive version of PTP1B (a mimic for an inhibited version of PTP1B) enhances luminescence. FIG. 29B shows one embodiment of Operon A in which the GOI is a gene for spectinomycin resistance (SpecR). PTP1B inhibits growth on spectinomycin, while a catalytically inactive version of PTP1B (a mimic for an inhibited version of PTP1B) enhances growth. The MidT substrate is used herein.

[0079] FIG. 30A-B illustrates Optimization of operon.

[0080] FIG. 30A shows one embodiment of operon from A in which the GOI is a bacterial luciferase (LuxAB), the PTP1B is missing, and the substrate is a peptide from Kras, midT, ShcA, or EGFR. Although all substrates can be phosphorylated by Src kinase, only two substrates bind to the SH2 domain tightly enough to enable significant luminescence over background (0% arabinose). FIG. 30B shows one embodiment of operon from A (here, contained on a single plasmid) in which the GOI is a bacterial luciferase (LuxAB) and PTP1B is missing. The Y/F mutation on the substrate domain (blue) prevents it from being phosphorylated. The RBS sites toggle expression of the Src kinase.

[0081] FIG. 31A-D illustrates Applications of operons.

[0082] FIG. 31A illustrates an exemplary conceptualization of a screen for microbially synthesizable inhibitors of PTP1B. When transformed with one embodiment of Operon A (or operon B), a cell capable of synthesizing PTP1B-inhibiting metabolites will produce a different GOI output than a cell that does not produce such metabolites. Because abietane-type diterpenoids can both (i) inhibit PTP1B and (ii) be synthesized in E. coli, we believe that a strain of E. coli that contains both Operon A and a pathway for building abietane-type diterpenoids could be evolved to build inhibitors of PTP1B. Here, the GOI could be a gene for luminescence or fluorescence (low throughput) or antibiotic resistance (high throughput). FIG. 31B illustrates an exemplary conceptualization of a screen for photoswitchable enzymes. Consider a fusion of PTP1B to LOV2 or BphP1 (here, the highlighted helices show N-terminal connection points on these two proteins). For this example, illumination of the PTP1B-LOV2 with 455 nm light reduces its activity; illumination of the PTP1B-BphP1 fusion with 650 nm light reduces its activity, while illumination of the PTP1B-BphP1 fusion with 750 nm light enhances its activity. When transformed with operon C (which would contain one of these fusions), a cell will produce a different GOI output under different illumination conditions. FIG. 31C illustrates an exemplary conceptualization of a screen for selective mutants of enzymes. When transformed with a version of operon B where (i) PTP1B is also active on the decoy and (ii) the second PTP (TC-PTP in our example) is missing, a cell containing a mutant of PTP1B will most effectively transcribe the GOI when PTP1B is only active on the decoy substrate. FIG. 31D illustrates an exemplary conceptualization of a screen for selective substrates. When transformed with a version of operon B where (i) the decoy is missing, (ii) the first enzyme (PTP1B in our example) is under an inducible promoter, (iii) a second PTP (TC-PTP in our example) is under a second inducible promoter, and (iv) the GOI includes a gene for antibiotic resistance and a gene that produces a toxic product in the presence of a non-essential substrate, a cell containing a mutated substrate domain will grow under both condition 1 (inducer of PTP1B and non-essential substrate) and condition 2 (inducer of TC-PTP), when it binds to PTP1B, but not to TC-PTP.

[0083] FIG. 32A-B presents exemplary evidence of an evolutionarily conserved allosteric network. FIG. 32A refers to an exemplary results of a statistical coupling analysis. The orange and blue clusters represent two groups of interconnected residues, termed sectors, that exhibit strong intragroup correlations in nonrandom distributions of amino acids. The allosteric site (green inhibitor, PDB entry 1T4J), WPD loop (purple spheres), and active site (red inhibitor, 3EB1) are highlighted for reference. FIG. 32B refers to an exemplary analysis of crosstalk between pockets of PTP1B modeled with MD simulations. Pockets are represented as spheres, colored according to their persistency along the MD trajectory; the size of each sphere indicates its average volume in MD simulations. Links have thicknesses proportional to the frequency of inter-pocket merging and splitting events (i.e., communication). Two independent sets of interconnected pockets map closely to the sectors identified in SCA and, thus, suggest that these two sectors represent distinct domains of an evolutionarily conserved allosteric network. In the PTP1B-LOV2 fusions of FIG. 1, LOV2 modulates the activity of PTP1B by tapping into the allosteric network defined by sector A. Identification of sector A with a statistical coupling analysis of the PTP family thus indicates that the architecture for photocontrol described in FIG. 1 is broadly applicable to all protein tyrosine phosphatases.

[0084] FIG. 33A-E illustrates an embodiment of a genetically encoded system that links the activity of an enzyme to the expression of a gene of interest (GOI). Error bars in FIG. 33B-E denote standard deviation with n=3 biological replicates.

[0085] FIG. 33A illustrates an embodiment of a bacterial two-hybrid system that detects phosphorylation-dependent protein-protein interactions. Components include (i) a substrate domain fused to the omega subunit of RNA polymerase (yellow), (ii) an SH2 domain fused to the 434 phage cI repressor (light blue), (iii) an operator for 434cI (dark green), (iv) a binding site for RNA polymerase (purple), (v) Src kinase, and (vi) PTP1B. Src-catalyzed phosphorylation of the substrate domain enables a substrate-SH2 interaction that activates transcription of a gene of interest (GOI, black). PTP1B-catalyzed dephosphorylation of the substrate domain prevents that interaction; inhibition of PTP1B re-enables it. FIG. 33B refers to an embodiment of the two-hybrid system from FIG. 33A that (i) lacks PTP1B and (ii) contains luxAB as the GOI. We used an inducible plasmid to increase expression of specific components; overexpression of Src enhanced luminescence. FIG. 33C refers to an embodiment of the two-hybrid system from FIG. 33A that (i) lacks both PTP1B and Src and (ii) includes a superbinder SH2 domain (SH2*, i.e., an SH2 domain with mutations that enhance its affinity for phosphopeptides), a variable substrate domain, and LuxAB as the GOI. We used an inducible plasmid to increase expression of Src; luminescence increased most prominently for p130cas and MidT, suggesting that Src acts on both substrate domains. FIG. 33D refers to an embodiment of a two-hybrid system from FIG. 33C with one of two substrates: p130cas or MidT. We used a second plasmid to overexpress either (i) Src and PTP1B or (ii) Src and an inactive variant of PTP1B (C215S). The difference in luminescence between systems containing PTP1B or PTP1B(C215S) was greatest for MidT, suggesting that PTP1B acts on this substrate. Right: An optimized version of the two-hybrid system (with bb030 as the RBS for PTP1B) appears for reference. FIG. 33E displays the results of an exemplary growth-coupled assay performed using an optimized B2H including SH2*, a midT substrate, optimized promoters and ribosome binding sites (bb034 for PTP1B), and SpecR as the GOI. This system is illustrated at the top of the figure. Exemplary growth results demonstrate that inactivation of PTP1B enables strain of E. coli harboring this system to survive at high concentrations of spectinomycin (>250 ?g/ml).

[0086] FIG. 34 illustrates exemplary experiments used to optimized the B2H system depicted in FIG. 33.

[0087] FIG. 35 illustrates exemplary experiments used to optimize the B2H system depicted in FIG. 33 for growth-coupled assays.

[0088] FIG. 36A-C depicts an exemplary metabolic pathway for the biosynthesis of terpenoids.

[0089] FIG. 36A depicts a plasmid-borne pathway for terpenoid biosynthesis: (i) pMBIS, which harbors the mevalonate-dependent isoprenoid pathway of S. cerevisiae, converts mevalonate to isopentyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP). (ii) pTS, which encodes a terpene synthase (TS) and, when necessary, a geranylgeranyl diphosphate synthase (GPPS), converts IPP and FPP to sesquiterpenes and/or diterpenes.

[0090] FIG. 36B depicts exemplary terpene synthases: amorphadiene synthase (ADS) from Artemisia annua, ?-humulene synthase (GHS) from Abies grandis, abietadiene synthase (ABS) from Abies grandis, and taxadiene synthase (TXS) from Taxus brevifolia.

[0091] FIG. 36C shows the results of an exemplary growth-coupled assay of strain of E. coli that contains both (i) an embodiment of the optimized bacterial two-hybrid (B2H) system (i.e., the B2H system from FIG. 33E) and (ii) an embodiment of a pathway for terpenoid biosynthesis (i.e., the pathway from FIG. 35A).

[0092] FIG. 37A-C provides an exemplary analysis of the inhibitory effects of terpenoids generated by different strains of E. coli.

[0093] FIG. 37A depicts the results of our analysis of the inhibitory effect of DMSO containing (i) no inhibitor and (ii) extracted compounds from the culture broth of the ADS-containing strain. FIG. 37B depicts the results of our analysis of the inhibitory effect of DMSO containing (i) extracted compounds from the culture broth of the GHS-containing strain (gHUM) or (ii) extracted compounds from the culture broth of the strain including the L450Y mutant of GHS. FIG. 37C depicts the results of our analysis of the inhibitory effect of DMSO containing (i) no inhibitor, (ii) extracted compounds from the culture broth of the ABS-containing strain, (iii) extracted compounds from the culture broth of the TXS-containing strain, and (iv) extracted compounds from the culture broth of the train strain containing a catalytically inactive variant of ABS.

[0094] FIG. 38 shows exemplary analysis of the product profiles of mutants of GHS that enabled growth in the presence of spectinomcyin.

[0095] FIG. 39 shows an analysis of an exemplary B2H systems that link the inhibition of other PTPs to cell survival.

[0096] FIG. 40A-E depicts exemplary embodiments of genetically encoded systems that link the activity of an enzyme to the expression of a gene of interest, and the application of those embodiments to (i) the prediction of resistance mutations, (ii) the construction of inhibitors that combat resistance mutations, and (ii) the evolution of inhibitors of kinases.

[0097] FIG. 40A depicts an exemplary first step in examining potential resistance mutations. By evolving a metabolic pathway to produce molecules that inhibit a known drug target (e.g., PTP1B); these molecules will permit expression of a gene of interest (GOI) that confers survival in the presence of a selection pressure (e.g., the presence of spectinomycin, an antibiotic). FIG. 40B depicts an exemplary second step in examining potential resistance mutations. In a second strain of E. coli, we will replace the original gene of interest with a second (GOI2) that confers conditional toxicity (e.g., SacB, which converts sucrose to levan, a toxic product); we will evolve the drug target to become resistant to the endogenous inhibitors, while still retaining its activity. This mutant will prevent expression of the toxic gene. FIG. 40C depicts an exemplary third step in combating resistance mutations. In a third strain of E. coli, we will evolve a metabolic pathway that produces molecules that inhibit the mutated drug target. In this way, we will both predictand, through our second evolved pathway, address-mutations that might cause resistance to terpenoid-based drugs. FIG. 40D depicts an exemplary genetically encoded system that detects inhibitors of an Src kinase. In brief, Src activity enables expression of a toxic gene (GOI2); inhibition of Src, in turn, would confer survival. FIG. 40E demonstrates one embodiment of a roof of principle for the B2H system describe in FIG. 40B. The system shown here includes two GOIs: SpecR and SacB. Expression of the GOIs confers survival in the presence of spectinomycin; expression of the GOIs causes toxicity in the presence of sucrose. The images depict the results of a growth-coupled assay performed on a strain of E. coli in the presence of various concentrations of sucrose. The strain harboring an active form of PTP1B (WT) grows better at high sucrose concentrations that the strain harboring an inactive form of PTP1B (C215S).

[0098] FIG. 41A depicts an exemplary strategy for the evolution of inhibitors of PTP1B.

[0099] FIG. 41A depicts an exemplary structural analysis used to identify targets for mutagenesis in the active sites of terpene synthases. It shows an alignment of the class I active site of ABS (gray, PDB entry 3s9v) and TXS (blue, PDB entry 3p5r) with the locations of sites targeted for site-saturation mutagenesis (SSM) highlighted on ABS (red). A substrate analogue (yellow) of TXS appears for reference. FIG. 41B depicts an exemplary strategy for introducing diversity into libraries of metabolic pathways: An iterative combination of SSM of key sites on a terpene synthase (as in a), error-prone PCR (ePCR) of the entire terpene synthase gene, SSM of sites on a terpene-functionalizing enzyme (e.g., P450), and ePCR of the entire terpene-functionalizing enzyme. FIG. 41C depicts an exemplary quantification the total terpenoids present in DMSO samples with extracts of various TS-containing strains. In brief, we performed site-saturation mutagenesis of six sites on ADS (analogous to the sites shown in FIG. 41A); we plated the SSM library on agar plates containing different concentrations of spectinomycin; we picked colonies that grew on a plate containing a high concentration (800 ?g/ml) of spectinomycin and used each colony to inoculate a separate culture; we used a hexane overlay to extract the terpenoids secreted into each culture broth; we dried the hexane extract in a rotary evaporator and re-suspended the solid in DMSO; and we used a GC-MS to quantify the total amount of terpenoids present in the DMSO. FIG. 41D depicts an exemplary analysis of the inhibitory effect of various extracts on PTP1B. In brief, the figure shows initial rates of PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) in the presence of terpenoids quantified in FIG. 41C. Two mutants of ADS (G439A and G400L) generate particularly potent inhibitors of PTP1B.

[0100] FIG. 42 depicts an exemplary analysis of the link between B2H activation and cell survival. An exemplary strain of E. coli that contains both (i) the optimized bacterial two-hybrid (B2H) system (FIG. 33E) and (ii) the terpenoid pathway depicted in FIG. 36A. Note: pTS includes GGPPS only when ABS or TXS are present; the Y/F operon corresponds to a B2H system in which the substrate domain cannot be phosphorylated. Survival at high concentrations of spectinomycin requires activation of the B2H system (i.e., phosphorylation of the substrate domain, a process facilitated by inhibition of PTP1B).

[0101] FIG. 43 provides exemplary product profiles of strains of E. coli harboring various terpene synthases. For this figure, the strain of E. coli harbored (i) the optimized B2H system (FIG. 33E) and (ii) the terpenoid pathway (FIG. 36A). The pathways corresponding to each profile differ only in the composition of the pTS plasmid, which contains TXS (taxadiene synthase from Taxus brevifolia and a geranylgeranyl diphosphate synthase from Taxus Canadensis); GHS (7-humulene synthase from Abies grandis); ADS (amorphadiene synthase from Artemisia annua); ABS (abietadiene synthase from Abies grandis and a geranylgeranyl diphosphate synthase from Taxus Canadensis); G400A (the G400A mutant of amorphadiene synthase from Artemisia annua); and G439L (the G439L mutant of amorphadiene synthase from Artemisia annua). Note that the two mutants of ADS yield different product profiles than the wild-type enzyme (ADS); our results indicate that products generated by these two mutants are more inhibitory than those generated by the wild-type enzyme (FIG. 41E).

[0102] FIG. 44A-D provides exemplary structural and sequence-based evidence that supports the extension the B2H system to other protein tyrosine phosphatases (PTPs).

[0103] FIG. 44A provides an exemplary structural alignment PTP1B and PTPN6, two PTPs that are compatible with the B2H system (see FIGS. 1e and 7 of Update A for evidence of compatibility). We used the align function of PyMol to align each structure of PTPN6 with either (i) the ligand-free (3A5J) or (ii) ligand-bound (2F71) structure of the catalytic domain of PTP1B. The align function carries out a sequence alignment followed by a structural superposition and, thus, effectively aligns the catalytic domains of both proteins. FIG. 44B provides an exemplary structural comparison of PTP1B and PTPN6; the root-mean-square deviations (RMSD) of aligned structures of PTP1B and PTPN6 range from 0.75 to 0.94 ?. FIG. 44C proves an exemplary sequence alignment of the catalytic domains of PTP1B (SEQ ID NO: 3) and PTPN6 (SEQ ID NO: 4) (EMBOSS Needle.sup.1). FIG. 44D provides an exemplary sequence comparison of the catalytic domains of PTP1B and TPPN6. The sequences share 34.1% sequence identity and 53.5% sequence similarity. In summary, the results of this figure indicate that our B2H system can be readily extended to PTPs that possess catalytic domains that are (i) structurally similar to the catalytic domain of PTP1B (here, we define structural similarity as two structures that when aligned, have with an RMSD of ?0.94 ? RMSD with the framework similar to the one used by the align function of PyMol) and/or (ii) sequence similar to the catalytic domain of PTP1B (here, we define sequence similarity as ?34% sequence identity or ?53.5% sequence similarity as defined by the EMBOSS Needle algorithm).

DEFINITIONS

[0104] As used herein, the use of the term operon may refer to a cluster of genes under the control of a single promoter (as in a classical definition of an operon) and may also refer to a genetically encoded system comprising multiple operons (e.g., the bacterial two-hybrid system).

[0105] As used herein, phosphorylation-regulating enzymes refer to proteins that regulate phosphorylation.

[0106] As used herein, phosphorylation refers to a biochemical process that involves the addition of phosphate to an organic compound.

[0107] As used herein, optogenetic actuator refers to a genetically encodable protein that undergoes light-induced changes in conformation.

[0108] As used herein, dynamic range refers to the ratio of activity in dark and light state (i.e., the initial rate in the dark/the initial rate in the presence of 455 nm light).

[0109] As used herein, operon refers to a unit made up of multiple genes that regulate other genes responsible for protein synthesis,

[0110] As used herein, operably linked refers to one or more genes (i.e. DNA sequences) suitably positioned and oriented in a DNA molecule for transcription to be initiated from the same promoter. DNA sequences that are operably linked to a promoter means that expression of the DNA sequence(s) is under transcriptional initiation regulation of the promoter.

[0111] As used herein, construct refers to an engineered molecule, e.g. ligated pieces of DNA as a DNA construct; a RNA construct as one contiguous sequence resulting from the expression of a DNA construct.

[0112] As used herein, fusion refers to an expressed product of an engineered construct i.e. a combination of several ligated sequences as one molecule or a single gene that encodes for a protein-protein fusion originally encoded by two genes.

[0113] As used herein, expression vector or expression construct refers to an operon, plasmid or virus designed for DNA expression of a construct in host cells, typically containing a promoter sequence operable within the host cell.

[0114] As used herein, promoter refers to a region of DNA that initiates transcription of a particular DNA sequence. Promoters are located near the transcription start sites of, towards the 5 region of the sense strand. Promoters may be constitutive promoters, such as cytomegalovirus (CMV) promoters in mammalian cells, or inducible promoters, such as tetracycline-inducible promoters in mammalian cells.

[0115] As used herein, transformation refers to a foreign nucleic acid sequence or plasmid delivery into a prokaryotic host cell, for example, an expression plasmid (e.g. a plasmid expression construct) inserted into or taken up by a host cell.

[0116] As used herein, transfection refers to the insertion of a nucleic acid sequence into a eukaryotic cell.

[0117] Transformation and transfection may be transient, such that the nucleic acid sequence or plasmid introduced into the host cell is not permanently incorporated into the cellular genome. A stable transformation and transfection refers to a host cell retaining the foreign nucleic acid sequence or plasmid for multip generations regardless of whether the nucleic acid or plasmid was integrated into the genome of the host cell.

[0118] As used herein, host in reference to a cell refers to a cell intended for receiving a nucelic acid sequence or plasmid or already harboring a a nucelic acid sequence or plasmid, eg. a bacterium.

[0119] As used herein, conjugate refers to a covalently attachment of at least two compounds, for example, a photosensing element attached to a phosphatase protein.

[0120] As used herein, decoy in reference to a protein construct that cannot bind to DNA and/or RNA polymerase.

DETAILED DESCRIPTION OF INVENTION

[0121] This invention relates to the field of genetic engineering. Specifically, the invention relates to the construction of operons to produce biologically active agents. For example, operons may be constructed to produce agents that control the function of biochemical pathway proteins (e.g., protein phosphatases, kinases and/or proteases). Such agents may include inhibitors and modulators that may be used in studying or controlling phosphatase function associated with abnormalities in a phosphatase pathway or expression level. Fusion proteins, such as light activated protein phosphatases, may be genetically encoded and expressed as photoswitchable phosphatases. Systems are provided for use in controlling phosphatase function within living cells or in identifying small molecule inhibitors/activator/modulator molecules of protein phosphatases associated with cell signaling.

[0122] The invention also relates to the assembly of genetically encoded systems (e.g., one or more operons) for detecting and/or constructing biologically active agents. For example, systems may be assembled in order to accomplish one or more goals, e.g. (i) to detect and/or synthesize small molecules that affect the activity of regulatory enzymes (e.g., protein phosphatases, kinases, and/or proteases); (ii) to detect and/or evolve regulatory enzymes modulated by light (e.g., light-responsive protein phosphatases, kinases, or proteases), etc. Small molecule modulators may include inhibitors of phosphatases known to be associated with human diseases or implicated with causing or perpetuating human diseases; activators of phosphatases implicated or known to be associated in human diseases (e.g., diabetes, obesity, and cancer); such small molecules may serve as chemical probes in studies of cell signaling; as structural starting points (i.e., leads); etc., for the development of pharmaceutical compounds for use in treating a human disease. Light-sensitive enzymes may include protein tyrosine phosphatases fused to optogenetic actuators (e.g., a LOV domain if phototropin 1). Such fusions could serve as tools for exerting spatiotemporal control over protein tyrosine phosphorylation in living cells

[0123] Further, microbial operons are provided that are designed for use in identifying either small molecule inhibitors, activators, or modulator molecules, photoswitchable enzymes, or biological components, including intracellularly expressed molecules, including, for examples, operons having components for use in whole cell microbial screening assay systems. Inhibitors/modulator molecules discovered using compositions, systems and methods described herein are contemplated for use in treating diseases such as diabetes, type II diabetes, obesity, cancer, and Alzheimer's disease, among other disorders associated with protein phosphatase enzymes.

[0124] In one embodiment, the present invention relates to a Protein tyrosine phosphatase 1B (PTP1B). PTP1B represents a valuable starting point for this study for four reasons: (i) It is implicated in diabetes.sup.5, obesity.sup.6, cancer.sup.30, anxiety.sup.31, inflammation.sup.32, the immune response.sup.7, and neural specification in embryonic stem cells.sup.33, (ii) The mechanisms underlying its subcellular localization are well understood (a short C-terminal anchor connects it to the ER; proteolysis of this anchor releases it to the cytosol).sup.2934. (iii) It can be expressed, purified, and assayed with ease.sup.35, (iv) It is a member of a class of structurally similar enzymes (PTPs) that could facilitate the rapid extension of architectures for making it photoswitchable. PTP1B represents both an experimentally tractable model system for testing strategies for optical control, and an enzyme for which optical modulation is contemplated to permit detailed analyses of a wide range of diseases and physiological processes.

[0125] Specifically related to exemplary Figures: FIGS. 1, 2, 3, 4, 8, 9, 12, and 13 describe optogenetic and imaging technologies (i.e., light-sensitive enzymes and genetically encodable biosensors) that could be evolved, improved, or optimized with the operon.; FIGS. 10 and 11 describe strategies for using the operon to evolve, improve, or optimize light-sensitive enzymes.; FIGS. 5, 6, 14, 15, 16, 17, 18, 19, 20, 28, 29, 30, and 31 support both (i) the development of an operon for detecting and/or evolving small molecules that inhibit known drug targets and (ii) the subsequent characterization of those molecules.; FIGS. 22, 23, 24, 25, 26, 27, and 32 provide examples of kinetic and biophysical characterizations of a microbially synthesizable molecule that inhibits PTP1B.

I. Protein Tyrosine Phosphatases (PTPs) and Protein Tyrosine Kinases (PTKs) in Relation to Disease.

[0126] Protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) are two classes of enzymes contributing to anomalous signaling events in a wide range of diseases (e.g., diabetes, cancer, atherosclerosis, and Alzheimer's disease, among others) and understanding disease progression.sup.14,36. Further, they are involved with regulating memory, fear, appetite, energy expenditure, and metabolism, thus use of such phosphorylation regulating enzymes may reveal links between seemingly disparate physiological processes.sup.14,22,13.

[0127] Embodiments for using light as photoswitchable constructs for controlling PTPs and PTKs is described herein. Accordingly, examples of photoswitchable constructs of PTPs and PTKs developed as described herein, should be broadly useful to biomedical researchers interested in understanding how healthy and diseased cells process chemical signals in addition to use for identifying specific alleles of PTPs and/or PTKs (i.e. gene sequences or proteins)or other enzymes that they regulate-linked to specific diseases, such as diabetes, etc., including subtypes of diseases, i.e. early onset, late onset, etc., and specific types of cancer, and for screening and testing molecules, including small molecules, for treating diseases associated with these alleles.

[0128] Although other references describe photocontrol of proteins, including using LOV2 conjugates, these references do not mention using phosphatases. Fan, et al., Optical Control Of Biological Processes By Light-Switchable Proteins. Wiley Interdiscip Rev Dev Biol. 4(5): 545-554. 2015. This reference describes blue light-oxygen-voltage-sensing (LOV) domains including the LOV2 C-terminal ?-helix, termed Ja, from Avena sativa phototropin. Linkage to the LOV domain can cage a protein of interest (POI), while light-induced conformational change in the LOV domain results in its uncaging. As one example, peptide kinase inhibitors can be caged by fusion to the C-terminus of LOV2. Exposure to light results in uncaging of the inhibitors for light modulating protein kinase activities in cells. WO2011133493. Allosteric regulation of kinase activity. Published Oct. 27, 2011. This reference describes fusion proteins comprising a kinase, including as examples, a tyrosine kinase (Src), a serine/threonine kinase (p38), and a ligand binding domain, e.g. a light-regulated LOV domain (where illumination is considered ligand binding), inserted in the N-terminal and/or C-terminal end or near the catalytic domain to produce allosteric regulation using a light-dependent kinase. Further, a LOV domain includes a LOV2 domain and/or J? domain from A. sativa phototropin I. WO2012111772 (A1) In Japanese with an English abstract. This reference abstract describes a polypeptide for the optical control of calcium signaling comprising an amino acid sequence including: a LOV2 domain composed of SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1. The construct has a LOV2 domain followed by a LOV2-Jalpha optical switch at the N terminus of the construct. U.S. Pat. No. 8,859,232. Genetically encoded photomanipulation of protein and peptide activity. Issued Oct. 14, 2014. This reference describes fusion proteins comprising protein light switches and methods of photomanipulating the activity of the fusion proteins to study protein function and analyze subcellular activity, as well as diagnostic and therapeutic methods. More specifically, a fusion protein comprising a protein of interest fused to a protein light switch comprising a light, oxygen or voltage (LOV2) domain of Avena sativa (oat) phototropin 1, wherein illumination of the fusion protein activates or inactivates the protein of interest. The protein of interest is a functional domain of a human protein. As an example, a LOV2-J? sequence of phototropin1 (404-547) was fused to the N-terminus of RacI so that the LOV domain in its closed conformation would reversibly block the binding of effectors to RacI.

A. Protein Tyrosine Phosphatases (PTPs).

[0129] Protein tyrosine phosphatases (PTPs) are a class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevantand, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase IB (PTP IB) with large-scale bioinformatic analyses to examine allosteric communication in PTPs. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP IB possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from kinetic studies show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations.

[0130] In one embodiment, a tyrosine phosphatase and photosensitive protein as described herein may be attached to a drug for use in medical treatments. In contrast to EP2116263, Reversibly light-switchable drug-conjugates. Published Nov. 11, 2009 which does not mention tyrosine phosphatase, and which describes photoswitchable conjugates of protein phosphatase calcineurin attached to a photoisomerizable group B and also attached to a drug for use in medical treatments (neither of these groups are genetically encodable). As one example in EP2116263, tumor growth is suppressed by inhibition of the protein phosphatase calcineurin. A photoisomerizable group B, for near UV (e.g. 370 nm) or near IR (e.g. 740 nm) induced activity, does not include a light responsive plant protein phototropin 1 LOV2 N-terminal alpha helix.

[0131] Receptor PTPs are contemplated for conjugation to light sensing proteins, as described herein. In contrast, Karunarathne, et al., Subcellular optogeneticscontrolling signaling and single-cell behavior. J Cell Sci. 128(1):15-25, 2015, describes photosensitive domains, such as bacteria light-oxygen-voltage-sensing (LOV and LOV2) domains including a C-terminal helical J? region, tagged to receptor tyrosine kinases (RTKs), there were no specific examples, there was no mention of a tyrosine phosphatase nor a plant phototropin 1 LOV2 N-terminal alpha helix. Optical activation of an inositol 5-phosphatase was shown, but inositol 5-phosphatase is not a protein phosphatase.

B. Enzymatic Phosphorylation of Tyrosine Residues.

[0132] Enzymatic phosphorylation of tyrosine residues has a role in cellular function and is anomalously regulated in an enormous range of diseases (e.g., diabetes, cancer, autoimmune disorders, and Noonan syndrome. It is controlled by the concerted action of two classes of structurally flexibleand dynamically regulatable-enzymes: protein tyrosine kinases (PTKs), which catalyze the ATP-dependent phosphorylation of tyrosine residues, and protein tyrosine phosphatases (PTPs), which catalyze the hydrolytic dephosphorylation of phosphotyrosines (5, 6). A detailed understanding of the mechanisms by which these enzymes respond to activity-modulating structural perturbations (i.e., mutations, post-translational modifications, or binding events) can, thus, illuminate their contributions to various diseases and facilitate the design of new PTK- or PTP-targeted therapeutics.

[0133] Over the last several decades, many biophysical studies have dissected the catalytic mechanisms and regulatory functions of PTKs (7, 8), which are common targets of pharmaceuticals.(9) Detailed analyses of PTPs, by contrast, have lagged behind.(10) These enzymes represent an underdeveloped source of biomedical insight and therapeutic potential (no inhibitors of PTPs have cleared clinical trials); they are, thus, the focus of this study.

[0134] PTPs uses two loops to dephosphorylate tyrosine residues. The eight-residue P-loop binds phosphate moieties through a positively charged arginine, which enables nucleophilic attack by a nearby cysteine, and the ten-residue WPD loop contains a general acid catalystan aspartatethat protonates the tyrosine leaving group and hydrolyzes the phosphoenzyme intermediate{circumflex over ()} 11-13) During catalysis, the P-loop remains fixed, while the WPD loop moves ?10A between open and closed states; nuclear magnetic resonance (NMR) analyses suggest this movement controls the rate of catalysis.(14)

[0135] Recent analyses of protein tyrosine phosphatase IB (PTP IB) a drug target for the treatment of diabetes, obesity, and breast cancer, indicate that motions of its WPD loop are regulated by an allosteric network that extends to its C-terminus (FIG. 1B) (15, 16). This network is susceptible to modulation by both (i) inhibitors that displace its C-terminal a7 helix (17, 18) and (ii) mutations that disrupt communication between the a(alpha)7 helix and the WPD loop (15); the specific collection of residues that enable allosteric communication in PTP1B and other PTPs has yet to be fully resolved.

[0136] Protein tyrosine phosphatase 1B (PTP1B). PTP1B represents a valuable tool for use in identifying potential therapeutics for at least four reasons: (i) It is implicated in diabetes.sup.5, obesity.sup.6, cancer.sup.30, anxiety.sup.31, inflammation 2, the immune response.sup.7, and neural specification in embryonic stem cells.sup.33, (ii) The mechanisms underlying its subcellular localization are well understood (a short C-terminal anchor connects it to the ER; proteolysis of this anchor releases it to the cytosol).sup.2934. (iii) It can be expressed, purified, and assayed with ease.sup.35, (iv) It is a member of a class of structurally similar enzymes (PTPs) that could facilitate the rapid extension of architectures for making it photoswitchable. PTP1B, thus, represents both an experimentally tractable model system for testing strategies for optical control, and an enzyme for which optical modulation will permit detailed analyses of a wide range of diseases and physiological processes.

[0137] Spatial regulation and intracellular signaling. PTP1B demonstrates, by example, the value of photoswitchable enzymes for studying spatial regulation in intracellular signaling. It is hypothesized to inactivate receptor tyrosine kinases through (i) contacts between endosomes and the ER.sup.37,38, (ii) contacts between the plasma membrane and extended regions of the ER.sup.39, and (iii) direct protein-protein interactions enabled by its partial proteolysis and release into the cytosol.sup.34. The role of different mechanisms (or locations) of PTP1B-substrate interaction in determining the outcomes of those interactions is poorly understood. Evidence suggesting a relationship between the location of PTP1B and its role in signaling has arisen in studies of tumorigenesis. Inhibition of PTP1B can suppress tumor growth and metastasis in breast.sup.30,40, lung.sup.3,41, colorectal.sup.9, and prostate cancers,.sup.42,43 while its upregulation has similar effects in lymphoma.sup.3,41. Recent evidence suggest that the former effect may result from inhibition of cytosolic PTP1B.sup.45; the cause of the latter is unclear. At present, there are no tools to investigate the differential influence of spatially distinct subpopulations of PTP1B on tumor-associated signaling events within the same cell. Photoswitchable variants of PTP1B represent such a tool.

[0138] Network biology. Signaling networks are often represented as nodes (proteins) connected by lines (interactions).sup.46. Such maps capture the connectivity of biochemical relay systems, but obscure spatial context the ability of a single interaction to occur in multiple locations and, perhaps, to stimulate multiple signaling outcomes. This study develops a set of tools that will enable detailed studies of the role of spatial context in guiding the propagation of signals through biochemical networks; such an examination contributes to understanding the role of PTP1B in cell signaling (and processes associated with tumorigenesis), and generally relevant to the study of any enzyme that exists in spatially distinct subpopulations within the cell.

II. Optogenetic Actuators

[0139] Optogenetic actuators (genetically encodable proteins that undergo light-induced changes in conformation) provide a convenient means of placing biochemical events under optical control. Alone, or when fused to other proteins, they have enabled optical manipulation of biomolecular transport, binding, and catalysis with millisecond and submicron resolution in living cells. Our approach addresses two major deficiencies in existing technologies: Observational interference and illuminating half the story. Existing strategies to control the activity of enzymes with light interfere with native patterns of protein production, localization, and interaction (often by design) and, thus, make direct interrogation and/or control of those patterns-which determine how biochemical signals are processed-difficult. There are several methods to control protein kinases with light, but no analogous methods for controlling protein phosphatases. As signaling networks are regulated by the concerted action of both classes of enzyme, comprehensive control and/or detailed dissections of those networks require methods for controlling both.

[0140] Embodiments described herein comprise (i) an approach for controlling the activity of proteins with light without disrupting their wild-type activities and (ii) a demonstration of this approach on a protein of particular importance: protein tyrosine phosphatase 1B (PTP1B), a regulator of cell signaling and a validated drug target for the treatment of diabetes, obesity, and cancer. There are no known photoswitchable protein tyrosine phosphatases. The PTP1B-LOV2 construct reported in this filing is the first. (ii) The N-terminal alpha helix of LOV2 is ignored in most studies (even reviews of optical switches) and has not been used as an exclusive connection point for optical modulation of enzymes.

[0141] We have developed a photoswitchable version of PTP1B by fusing the C-terminal allosteric domain of this enzyme to the N-terminal alpha helix of a protein light switch (i.e., the LOV2 domain of phototropin 1 from Avena sativa). We present evidence that this general architecturewhich is unique in the placement of LOV2 away from the active site of PTP1B (minimally disruptive)can be extended to other PTPs and, perhaps, PTKs. For example, we used a statistical coupling analysis to show that the allosteric network exploited in our PTP1B design is preserved across the PTP family.

[0142] Alone, or when fused to other proteins, optogenetic actuators have enabled optical manipulation of biomolecular transport, binding, and catalysis with millisecond and submicron resolution.sup.15,16. At least three deficiencies limit their use in detailed studies of signaling networks: Observational interference. Existing strategies to control the activity of enzymes with light interfere with native patterns of protein production, localization, and interaction.sup.16,17 (often by design) and, thus, make direct interrogation of those patterns-which determine how biochemical signals are processed.sup.10 difficult. Illuminating half the story. There are several methods to control protein kinases with light.sup.18,19, but no analogous methods for controlling protein phosphatases. As signaling networks are regulated by the concerted action of both classes of enzyme, detailed dissections of those networks require methods for controlling both. A limited palette of actuators. Optogenetic actuators that enable subcellular control of enzyme activity require the use of blue or green light.sup.15. These wavelengths exhibit significant phototoxicity.sup.20, suffer from short biological penetration depths.sup.21, and, as a result of their spectral similarity, limit actuation to individual signaling events, rather than multiple events simultaneously.

A. Photoswitchable Constructs: Advantages Over Other Exemplary Technologies.

[0143] As described herein, a photoswitch describes a protein-protein architecture (e.g., a PTP1B-LOV2 fusion) that is optically active in its monomeric form. A reference, WO2013016693. Near-infrared light-activated proteins. Publication Date Jan. 31, 2013, relies on homodimerization. In contrast, optical control as described herein is over a larger range of proteins, including both those that require homodimerization and those that do not, unlike in WO2013016693. Further, this reference describes types of photosensory modules including blue light-sensitive flavoproteins found in plants; photoreceptors of blue-light using flavin adenine dinucleotide (BLUF); Light, Oxygen, or Voltage sensing (LOV) types, which includes plant and bacterial photoreceptors; and plant/microbe phytochromes, sensitive to light, i.e. light-induced helix rotation in the red-to-NIR region. More specifically described with examples are bacteriophytochrome (Bph)-based photoactivated fusion proteins, using light-responsive alpha helixes from Rhodobacter sphaeroides (BphG) fused to proteins such as protein phosphatases, protein kinases, membrane receptors, etc. E. coli, are modified so as to exhibit the level of photoactivity of these expressed fusion proteins, i.e. in the presence or absence of red-to-NIR light. Although blue color changes in E. coli expressing fusion proteins are described in response to light, these blue bacteria are the result of using far-red/NIR-light for photoactivating a fusion protein that in turn activates lacZ expression in the presence of Xgal, not a photoresponse to exposure to blue light. However, there is no specific mention of a tyrosine phosphatase or a plant phototropin 1 LOV2 N-terminal alpha helix. In fact, reviews on optogenetics tend to depict LOV2 as having one terminal helix: The C-terminal Jalpha helix. While there are studies/patents indicating that simple insertion of the LOV2 domain enables photocontrol they rely on the underlying assumption that the Jalpha helix is unwinding to produce the controlling effect, not the A alpha helix as described herein.

B. A Cage-Free Approach to Control Protein Tyrosine Phosphatases and Protein Tyrosine Kinases with Light.

[0144] Current strategies for using light to control the activity of enzymes (as opposed to their concentration or location) rely on cage-based systems: a light-responsive protein, when fused to an enzyme of interest, controls access to its active site.sup.16,17. Unfortunately, such architectures can alter the affinity of enzymes for binding partners and change their susceptibility to activity modulating modifications (e.g., phosphorylation).sup.16,18. These effects complicate the use of optogenetics to study signaling. This study will develop a cage-free, allostery-based approach for optical control that minimizes interference between enzymes and their substrates (and other binding partners). This approach will help preserve native patterns of protein localization, interaction, and post-translational modification and, thus, facilitate studies of the influence of those patterns on intracellular signaling.

[0145] 2. A genetically encoded photoswitchable phosphatase. There are no genetically encodable photoswitchable phosphatases; the chimeras developed in this proposal will be the first. Photoswitchable variants of PTP1B will enable detailed studies of a wide range of interesting PTP1 B-regulated processes (e.g., insulin, endocannabinoid, and epidermal growth factor signaling.sup.49,51, and cell adhesion and migration.sup.52). Photoswitchable phosphatases, in general, will provide a useful class of tools for studying cell biology (particularly in concert with photoswitchable kinases, which could enable complementation experiments).

[0146] Hypothesis: The catalytic domains of PTPs and PTKs possess C-terminal ?-helices that are distal to their active sites, yet capable of modulating their catalytic activities (for at least a subset of enzymes the generality of this function is not known).sup.23,24. We hypothesize that the fusion of this helix to the N-terminal ?-helix of the light-oxygen voltage 2 (LOV2) domain of phototropin 1 from Avena sativa-a photosensory domain with terminal helices that unwind in response to blue light.sup.2526will yield enzyme-LOV2 chimeras that exhibit light-dependent catalytic activities, yet retain their native substrate specificities and binding affinities.

[0147] Experimental approach: We will attach the C-terminal ?-helix of PTP1B to the N-terminal ?-helix of LOV2 at homologous crossover points, and we will assess the influence of photoactivation on the catalytic activity of the resulting chimeras. This effort will involve the use of (i) kinetic assays and binding studies to characterize the substrate specificities and binding affinities of photoswitchable constructs and (ii) crystallographic and spectroscopic analyses to examine the structural basis of photocontrol. Informed by these studies, we will extend our approach to striatal-enriched protein tyrosine phosphatase (STEP) and protein tyrosine kinase 6 (PTK6), enzymes implicated in Alzheimer's disease and triple-negative breast cancer, respectively.

[0148] We will combine sophisticated biophysical studies, synthetic biology, and fluorescence microscopy to (i) develop protein architectures that enable optical control of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) without interfering with their wild-type activities or binding specificities, (ii) evolve PTPs and PTKs modulated by red light, and (iii) develop an imaging methodology to study spatially localized signaling events in living cells.

[0149] We will begin our study with PTP1B, a validated drug target for the treatment of diabetes, obesity, and breast cancer, and an enzyme for which optogenetic tools will be particularly useful to address current gaps in knowledge (e.g., the role of spatially distinct subpopulations of PTP1B in promoting or suppressing the growth of tumors.sup.22). Using it as a model, we will establish the generality of our methods by extending them to other PTPs and PTKs.

C. A Photoswitchable Variant of PTP1B.

[0150] Our first objective seeks to use LOV2, a protein with terminal helices that unwind in response to blue light, to control the activity of PTP1B, an enzyme for which unwinding of the C-terminal ?-helix disrupts activity by distorting its catalytically essential WPD loop (FIG. 1AB, FIG. 6). To assess the feasibility of this goal, we constructed five PTP1B-LOV2 chimeras (joined at homologous crossover points): three chimeras showed light-dependent catalytic activity on 4-methylumbelliferyl phosphate (4M) (FIG. 1G). A subsequent mutational analysis of one chimera indicated that mutations in the ?-helix that links PTP1B to LOV2 can improve catalytic activity and dynamic range (DR, the ratio of dark/light activities; FIG. 1G). Our ability to buildand begin optimizinga photoswitchable PTP1B-LOV2 chimera by screening a small number of constructs suggests that rational design will allow us to build a chimera sufficient for intracellular signaling studies. We note: Our most photoswitchable chimera has a DR of 2.2; previous imaging studies suggest that a DR of 3-10 is sufficient to control intracellular signaling.sup.218,19.

[0151] More specifically, FIG. 1C demonstrates some of differences over other types of optical control. The y-axis of the top plot indicates the activity of each construct in the dark (i.e., the initial rate of PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate); the y-axis of the bottom plot indicates the ratio of activity in dark and light state (i.e., the initial rate in the dark/the initial rate in the presence of 455 nm light), i.e. dynamic range.

[0152] Black bars show the activity and dynamic range for a set of eight initial constructs that differ in the crossover point (see the bottom of FIG. 1B). Some of these constructs are photoswitchable, and some are not. Version 7 shows the greatest photoswitchabilitythe dynamic range is approximately 1.8.

[0153] More specifically, colors are associated with different types of constructs. Black: different crossover point (see FIG. 1B for crossover points); Gray: different partitioning of the linker (see, Linker section below); Light blue: the Jalpha helixthis is at the C-terminus of the LOV2 domain; Dark blue: the Aalpha helixthis is at the N-terminus of the LOV2 domain and, thus, on the region that links it to PTP1B; Yellow: the alpha7 helix of PTP1Bthis is at the C-terminus of PTP1B and, thus, on the region that links PTP1B to LOV2; Orange: combination: a combination of sites from the previous colors, see below for additional information.

[0154] These results were surprising, in part, because a recent review on optogenetics shows that that photocontrol of activity requires the J? helix of LOV2, where J? is a C-terminal helix which resides in a folded state against the LOV domain core, to be attached to a protein of interest, see Repina, N. A., Rosenbloom, A., Mukherjee, A., Schaffer, D. V. & Kane, R. S. At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior. Annu. Rev. Chem. Biomol. Eng. 8, 13-39 (2017). Photoactivation with blue light converts the noncovalent interaction between the LOV core and its bound flavin chromophore, FMN, into a covalent one through a conserved cysteine residue. The accompanying light-induced conformational change displaces the J? helix away from the protein core, leading to uncaging of a fused effector domain (e.g., the kinase domain of phot1). J? helix reverts to its dark-state caged conformation within minutes owing to spontaneous decay of the protein-cofactor bond.

[0155] Several limitations of the native AsLOV2 domain have motivated efforts to engineer improved variants. First, when fused to foreign protein domains, spontaneous undocking of the J? helix can lead to a relatively high dark-state activity, resulting in a low dynamic range upon AsLOV2 uncaging (26). For example, the light-inducible DNA-binding system LovTAP has only a fivefold change in DNA affinity between the dark and illuminated states (27). To address this issue, Strickland et al. (26) used rational design to introduce four mutations into AsLOV2 that stabilized the docking of J? to the LOV core. This increased the dynamic range of LovTAP from 5-fold to 70-fold, an approach that can be applied to other LOV domain optogenetic systems to reduce dark-state activity. AsLOV2 fusions are also particularly sensitive to linker lengths and the size and structure of attached domains (28, 29), and as a result, each new fusion protein switch requires optimization to achieve low dark-state and high light-state activity in mammalian cells.

[0156] In contrast to the J? helix-protein chimers, as shown herein, the A? helix not the J? helix is attached to the protein of interest to form photoswitchable constructs, e.g. PTPB1.

Exemplary Linkers.

[0157] Gray bars of FIG. 1C show the activity and dynamic range of mutants of version 7 in which the linker has been re-partitioned. In other words, version 7 has the following linker region: LSHEDLATTL (SEQ ID NO: 5), where the underlined region LSHED (SEQ ID NO: 6) corresponds to the C-terminus of PTP1B, and the region LATTL (SEQ ID NO: 7) corresponds to the N-terminus of LOV2. Version 7.1 has sequence LSHEDATTL (SEQ ID NO: 8); version 7.2 has sequence LSHEDTTL (SEQ ID NO: 9), and so on. Here, we find that version 7.1 has the same dynamic range as version 7, but a higher activity. We, thus, used version 7.1 for further optimization.

Exemplary Mutations.

[0158] Light blue bars show the activity and dynamic range of mutants of version 7.1 in which the J? helix contains helix-stabilizing mutations. Curiously, these improve the activity of 7.1, but do not improve its dynamic range.

[0159] Dark blue bars show the activity and dynamic range for mutants of version 7.1 in which the A? helix contains helix-stabilizing mutations. One of these mutations (T406A) improves dynamic range; we used this version for further studies.

[0160] Yellow bars show the activity and dynamic range of mutants of version 7.1 in which the ?7 of PTP1B has helix-stabilizing mutations; the orange bars show the activity and dynamic range for mutants of version 7.1 in which the multiple mutations are combined. Neither of the constructs associated with yellow and orange bars show improved characteristics of 7.1 (T406A).

[0161] A minimally disruptive approach. Two kinetic studies indicate that our architecture for photocontrol does not interfere with the native substrate specificity or binding behavior of PTP1B: (i) An analysis of the activity of chimera E3 (from FIG. 1D) on p-nitrophenyl phosphate (pN) indicates that light affects k.sub.cat, but not K.sub.m (FIGS. 2K and L). (ii) An analysis of activities on three substrates of different sizes (4M, pN, and a peptide) shows that DR is the same for all three (FIG. 2L-K). The results of both studies are consistent with our hypothesized mechanism of photocontrol: LOV2-induced unwinding of the C-terminal ?-helix of PTP1B disrupts the movement of its catalytically essential WPD loop, which controls the rate of catalysis, but has little influence on substrate binding affinity.

[0162] Biophysical studies. Photoswitchable chimeras express at titers (?100 mg/L) sufficient to carry out detailed biophysical analyses. We performed a preliminary set of these analyses on chimera E3. (i) We use circular dichroism (CD) to examine the influence of photoactivation on its secondary structure; spectral measurements indicate that photoactivation reduces ?-helical content (222 nm; FIG. 2B). (ii) We used the amplitude at 222 nm to measure a post-activation recovery time for ?-helical content: T.sub.r?30 s (FIG. 2E). This value is similar to the recovery times of previously developed LOV2-based photoswitchable constructs, (iii) We used tryptophan fluorescence to measure a post-activation recovery time of tryptophan residues: T.sub.r-50 s (FIG. 2F). Tryptophan fluorescence is a rough metric for the conformation of PTP1B (which has seven tryptophan residues, compared to one in LOV2); this slower recovery time, thus, suggests that PTP1B takes longer than LOV2 to refold, (iv) We identified a set of crystallization conditions (those previously used to crystallize PTP1 B.sub.WT) to grow crystals of E3 (FIG. 2F). (V) We collected a two-dimensional .sup.1H-.sup.15N HSQC spectrum of PTP1B.sub.WT, and assigned ?65% of non-proline peaks. These NMR experiments, which are recent, have yet to include PTP1B-LOV2 chimeras; but the ease with which we carried them out (a single try) suggests that similar analyses of chimeras will be straightforward. The experimental tractability of PTP1B-LOV2 chimeras will enable a comprehensive biophysical analysis of variants with different photophysical properties.

[0163] Example 1. To develop a cage-free approach to control protein tyrosine phosphatases and kinases with light. This section develops an approach for placing enzymes under optical control without disrupting their native interactions. We will demonstrate this approach with PTP1B and, then, extend it to STEP and PTK6. We will know that we are successful when we have a PTP1B-LOV2 chimera that exhibits a three- to ten-fold change in activity between light and dark states, and when we have identified structure-based design rules that facilitate fine-tuning of the photophysical properties of photoswitchable variants of PTP1B, STEP, and PTK6.

D. Development of a Photoswitchable Variant of PTP1B.

[0164] The efforts in this section assumeand with crystallographic, kinetic, and binding studies, attempt to confirmthat optogenetic actuation systems located far from active sites are less likely to disrupt wild-type behaviors that actuation systems located nearby. Kinetic studies of preliminary PTP1B-LOV2 chimeras (i.e., chimeras in which the C-terminal helix of PTP1B is connected to the N-terminal helix of the LOV2 domain of phototropin 1 from A vena sativa) support this hypothesis: light inhibits their activity by affecting k.sub.cat, not K.sub.m, and they show wild-type kinetics on 4-methylumbelliferyl phosphate (4M), a model substrate (FIG. 1G and FIG. 2K). Photomodulation of k.sub.cat, but not K.sub.m suggests that LOV2 exploits an allosteric network to distort the WPD loop (FIG. 6).

[0165] Our initial constructs, which represent the first reported examples photoswitchable protein phosphatases, will facilitate a systematic study of the functional advantages of different chimera architectures. We are particularly interested in understanding how (i) the length of the linker that connects PTP1B and LOV2 and (ii) the stability of the terminal helices of LOV2 affect catalytic activity and dynamic range. We will study these relationships by combing spectroscopic analyses with kinetic studies. Spectroscopic analyses will show how different PTP1B-LOV2 chimeras rearrange under illumination (e.g., we will use CD and fluorescence spectroscopy to measure photomodulation of ?-helical content and tryptophan fluorescence), and kinetic studies will reveal the influence of those rearrangements on catalytic activity and dynamic range.

[0166] The results of our biophysical analyses will facilitate the optimization of our chimera for in vitro cell studies. We will target a chimerahereafter, referred to as PTP1B.sub.PSwith the following properties: a dynamic range (DR) of 3-10, a recovery time of T.sub.r?15-60 s, and wild-type activity (in its activated state). Previous optogenetic studies suggest that these attributes enable optical control of cell signaling.sup.2,18,19. We note: Biophysical studies of PTP1B indicate that the removal of its C-terminal ?-helix can reduce its activity by a factor of four.sup.57; accordingly, we believe that LOV2 can modulate the activity of PTP1B by at least fourfold (of course, LOV2 may trigger structural distortions more pronounced than those of a simple truncation).

E. Characterization of PTP1B-Substrate and PTP1B-Protein Interactions.

[0167] We will assess the influence of LOV2 on the substrate specificity of PTP1B by using kinetic analyses. Specifically, we will compare the activities of PTP1B.sub.WT and PTP1B.sub.PS on three substrates: (i) p-nitrophenyl phosphate, a general substrate for tyrosine phosphatases, (ii) ETGTEEpYMKMDLG (SEQ ID NO: 10), a substrate of PTPs closely related to PTP1B, and (iii) RRLIEDAEpYAARG (SEQ ID NO: 11), a substrate specific to PTP1B. A comparison of values of k.sub.cat and K.sub.m on these substrates (FIG. 2K shows an example kinetic study) will reveal differences in the catalytic activities and specificities of PTP1B.sub.WT and PTP1B.sub.PS. These studies will also allow us to assess the substrate-dependence of photoswitchability (i.e., DR). Photomodulation is often assumed to be independent of substrate; there is, however, no biochemical basis for this assumption (particularly in cage-based systems, where substrates may bind with different affinities and, thus, have different abilities to compete with the caging protein). We will test it.

[0168] We will assess the ability of PTP1B.sub.PS to engage in the same protein-protein interactions as PTP1B.sub.WT by measuring the affinity of both enzymes for two native binding partners of PTP1B.sub.WT: LMO4 and Stat3. Binding isotherms based on changes in tryptophan fluorescence of PTP1B will facilitate this study (FIG. 7).

[0169] Our biochemical comparison of PTP1B.sub.WT and PTP1B.sub.PS may seem tedious, but we believe that this analysis is necessary to establish the relevance of future optogenetic observations to wild-type processes.

[0170] Biostructural characterization. We will investigate the structural basis of photocontrol in PTP1B.sub.PS by using X-ray crystallography and NMR spectroscopy. X-ray crystal structures will show how LOV2 affects the structure of PTP1B (and vice versa); NMR spectroscopy will show how LOV2 modulates catalytic activity. For crystallographic studies, we will crystallize PTP1B.sub.PS in its dark state (we will use the C450S mutation, which prevents formation of the cysteine adduct.sup.2,26) by screening crystallization conditions previously used for LOV2, PTP1B, and LOV2-protein chimeras (all of which have crystal structures.sup.2,35,58); preliminary results suggest that those used to grow crystals of PTP1B.sub.WT also yield crystals of PTP1B-LOV2 chimeras (FIG. 2J). For NMR studies, we will use heteronuclear single quantum coherence (HSQC) spectroscopy and transverse relaxation-optimized spectroscopy (TROSY) to monitor changes in the conformation and backboned dynamics of PTP1B.sub.PS before and after illumination. (We note: Backbone .sup.1H,.sup.13C, and .sup.15N chemical shifts have been assigned for PTP1B and LOV2.sup.59,60)

G. Exemplary Imaging Methodology to Study Subcellular Signaling Events in Living Cells.

[0171] This section uses PTP1B.sub.PS (a PTP1B-LOV2 chimera) to develop an approach for using confocal microscopy to probeand study-subcellular signaling events. We will know that this objective is successful when we can inactivate a within subcellular regions, monitor the effect of that inactivation with an FRET-based sensor, and isolate the contributions of different subpopulations of PTP1B (e.g., ER-bound and cytosolic) to sensor phosphorylation.

[0172] Hypothesis. The subcellular localization of PTPs and PTKs is controlled by domains proximal to their catalytic cores.sup.23,24. We hypothesize that the attachment of these domains to photoswitchable chimeras will give them wild-type localization patterns, and enable the use of confocal microscopy to study the contribution of spatially distinct subpopulations of PTPs and PTKs to cell signaling. Experimental approach: Within the cell, PTP1B exists in two spatially distinct subpopulations: attached to the cytosolic face of the endoplasmic reticulum, and free in the cytosola result of proteolysis of its short (?80 residue)C-terminal ER anchor.sup.29. We will (i) attach the ER anchor of wild-type PTP1B (PTP1 B.sub.WT) to our PTP1B-LOV2 chimera, (ii) compare the subcellular localization of the resulting chimera with that of PTP1B.sub.WT, (iii) use confocal microscopyin conjunction with a FRET-based sensor for phosphatase activityto control and monitor PTP1B activity within the cell, and (iv) develop a reaction-diffusion model to assess the contributions of spatially distinct subpopulations of PTP1B to changes in sensor phosphorylation over time and space. This work will yield a general approach for studying spatially localized signaling events in living cells.

Localization of PTP1B.SUB.PS..

[0173] To examine the localization of PTP1B.sub.PS in living cells, we will express three variants in COS-7 cells: (i) PTP1B.sub.PS_c45os, (ii) PTP1B.sub.PS_c45os attached to a short segment (?20 amino acids.sup.29) of the C-terminal ER anchor of PTP1B.sub.WT that contains only the transmembrane domain (but not the proteolysis site), and (iii) PTP1 B.sub.PS-c45os attached to the full C-terminal ER anchor of PTP1B.sub.WT (?80 amino acids.sup.29). We hypothesize that these constructs will have (i) cytosolic, (ii) ER-bound, and (iii) both cytosolic and ER-bound (i.e., wild-type) localization patterns, respectively. Using confocal microscopy, we will test this hypothesis by using the fluorescence of LOV2 to locate each chimera.sup.70. (In these studies, we will locate the ER with fluorescently-labeled SEC61B, an ER-associated transport complex.sup.71. The C450S mutation, which locks LOV2 in its fluorescent state, will prevent photoactivation during imaging).

[0174] COS-7 cells, fibroblast-like cells derived from the kidney tissue of the African green monkey, are particularly compatible with the aforementioned analysis for three reasons: (i) They are large and flat and, thus, facilitate imaging of spatially segregated subcellular regions.sup.72, (ii) They are compatible with commercially available transfection reagents.sup.73, (iii) Methods for inducing endocytosis.sup.71 and calpain expression.sup.74, two processes that influence the subcellular activity and localization of PTP1B, are well developed for these cells.

[0175] Control of PTP1B.sub.PS in living cells. We will examine the activity of PTP1B within subcellular regions by pairing confocal microscopy with a FRET-based sensor for protein phosphorylation (developed by the Umezawa group.sup.54; FIG. 12). This sensor will consist of a kinase substrate domain, a short flexible linker, and a phosphorylation recognition domain-all sandwiched between two fluorescent proteins (Clover, a green fluorescent protein, and mRuby2, a red fluorescent protein). Phosphorylation of the substrate domain will cause it to bind to the recognition domain, modulating (i.e., enhancing or reducing) FRET between the two fluorescent proteins. Our preliminary sensor, which uses substrate and SH2 domains compatible with PTP1B and src.sup.23,55 exhibits a 20% change in FRET in response to phosphorylation. We will attempt to optimize our sensor further by screening different substrate domains, SH2 domains, and linker lengths. Ouyang et al. built a FRET sensor for Src kinase activity that exhibits a ?120% change in FRET when phosphorylated.sup.55; we will use the architecture of this sensoror, perhaps the sensor itselfto inform our designs.

[0176] In our imaging experiments, we will use a 455-nm laser to inactivate PTP1B within subcellular regions (1-10 urn circles) and fluorescence lifetime imaging microscopy (FLIM) to monitor changes in sensor phosphorylation that result from that inactivation (FIG. 13). For these experiments, we will use siRNA to deplete PTP1 B.sub.WT and SEC61B to label the ER. The output will be a series of images in which the intensity of a pixel is proportional to the fluorescence lifetime of Clover (and, thus, the extent of sensor phosphorylation).

[0177] With this study, we are particularly interested in examining relationships between (i) the location of PTPIBps activation/inactivation, (ii) the size of the region of activation/inactivation, and (iii) the location and timing of changes in the phosphorylation state of the sensor. We will investigate these relationships by using a reaction-diffusion model. Equation 1 provides a simple example of a governing equation:

[00001]$\begin{array}{cc}\frac{?S_p\left(r,t\right)}{?t}=D_S{?}^2{S}_p+k_{\mathrm{cat}}^K\left[\mathrm{KS}\right]-k_{\mathrm{cat}}^p\left[{\mathrm{PS}}_p\right]-k_{\mathrm{on}}^K\left[P\right]\left[S_p\right]& \left(\mathrm{Eq}.1\right)\end{array}$

for the phosphorylated sensor (S.sub.P). Here, D.sub.s is the diffusion coefficient for the sensor; KS is the concentration of tyrosine kinase bound to unphosphorylated sensor; PS, is the concentration of PTP1B bound to phosphorylated sensor; P and S.sub.p are the concentrations of free PTP1B and free phosphorylated sensor, respectively; k{circumflex over ()}.sub.at and k{circumflex over ()}.sub.at are the catalytic constants for the tyrosine kinase and PTP1B, respectively; and k %.sub.n is the kinetic constant for sensor-PTP1B association. The kinase and phosphatase are assumed to bind only weakly with their products (an assumption that can be easily re-examined later). We may also supplement this model with tools such as BioNetGen, a web-based platform for generating biochemical reaction networks from user-specified rules for the mechanisms and locations of biomolecular interactions.sup.75; such a tool, which can accommodate cellular heterogeneity (e.g., organelles and other compartments), will help to support and expand our kinetic model.

[0178] We hypothesize that a version of our kinetic model in which the phosphatase diffuses freely will more accurately capture the phosphorylation state of the sensor (at a specified time and position from the irradiation region) in the presence of cytosolic PTP1B.sub.PS. By contrast, a version of the model in which phosphatase does not diffuse freely will more accurately capture the behavior of sensors in the presence of ER-bound PTP1B.sub.PS. Regression of either model against imaging data will enable estimation of the extent to which cytosolic and ER-bound PTP1B contribute to changes in sensor phosphorylation over time and space.

[0179] Image analysis. The ER exists as a vesicular network that is spread throughout the cell; inactivation of subcellular regions that are entirely ER or entirely cytosol is difficult. To enable analysis of spatially distinct subpopulations of PTP1B, we must, thus, estimate the amount of ER in different regions of irradiation. The discrepancy in length scales of ER heterogeneity (?20-100 pm) and irradiation (?1-10 pm) will permit such an estimation. We will work with two metrics: (i) the total fluorescence of labeled ER, and (ii) the anisotropy of labeled ER. Both metrics, by facilitating estimates of the populations of cytosolic and ER-bound PTP1B in an illuminated region, will help us to assess the contributions of those populations to changes in sensor phosphorylation.

Spatial Regulation and Intracellular Signaling.

[0180] PTP1B demonstrates, by example, the value of photoswitchable enzymes for studying spatial regulation in intracellular signaling. It is hypothesized to inactivate receptor tyrosine kinases through (i) contacts between endosomes and the ER.sup.37,38, (ii) contacts between the plasma membrane and extended regions of the ER.sup.39, and (iii) direct protein-protein interactions enabled by its partial proteolysis and release into the cytosol.sup.34. The role of different mechanisms (or locations) of PTP1B-substrate interaction in determining the outcomes of those interactions is poorly understood. Evidence suggesting a relationship between the location of PTP1B and its role in signaling has arisen in studies of tumorigenesis. Inhibition of PTP1B can suppress tumor growth and metastasis in breast.sup.30,40, lung.sup.0,41, colorectal.sup.9, and prostate cancers,.sup.42,43 while its upregulation has similar effects in lymphoma.sup.3-4.sup.4. Recent evidence suggests that the former effect may result from inhibition of cytosolic PTP1B.sup.45; the cause of the latter is unclear. At present, there are no tools to investigate the differential influence of spatially distinct subpopulations of PTP1B on tumor-associated signaling events within the same cell. Photoswitchable variants of PTP1B represent such a tool.

[0181] Network biology. Signaling networks are often represented as nodes (proteins) connected by lines (interactions).sup.46. Such maps capture the connectivity of biochemical relay systems, but obscure spatial contextthe ability of a single interaction to occur in multiple locations and, perhaps, to stimulate multiple signaling outcomes. This study develops a set of tools that will enable detailed studies of the role of spatial context in guiding the propagation of signals through biochemical networks; e.g. understanding the role of PTP1B in cell signaling (and processes associated with tumorigenesis), and generally relevant to the study of any enzyme that exists in spatially distinct subpopulations within the cells.

Generalization of Approach to Protein Tyrosine Phosphatases and Kinases.

[0182] Two observations suggest that our architecture for photocontrol (i.e., attachment of the N-terminus of LOV2 to the C-terminal ?-helix of an enzyme) is broadly applicable to PTPs and PTKs. (i) Structural alignments show that all PTPs possess, or, with a few mutations, can possessthe same allosteric communication network as PTP1B (FIG. 8A).sup.23. (ii) PTKs contain a C-terminal ?-helix that is distal to their active sites, yet capable of modulating their catalytic activities (FIG. 8B).sup.61.

[0183] We will assess the generalizability of our approach by building photoswitchable variants of striatal-enriched protein tyrosine phosphatase (STEP) and protein tyrosine kinase 6 (PTK6; FIG. 8A). STEP is a neuron-specific phosphatase that is overactive in several neurological disorders, prominently Alzheimer's disease, schizophrenia, and drug addiction.sup.62,63. PTK6, which may function orthogonally to PTP1B in some signaling pathways, is expressed in approximately 70% of triple-negative breast cancers and promotes metastasis.sup.50,64. Photoswitchable variants of STEP and PTK6, both of which exist in multiple spatially distinct subpopulations within cells.sup.50,62, will enable detailed studies of their intracellular signaling roles, which remain poorly characterized.

[0184] For STEP and PTK6, we will developand measure the substrate specificities of photoswitchable chimeras by using several kinetic assays. For STEP, we will use assays analogous to those employed with PTP1B. For PTK6, we will use the ADP-Glo kit developed by Promega, Inc..sup.65. This assay, which is compatible with any peptide substrate, converts ADP produced by PTK-catalyzed peptide phosphorylation to a luminescent signal. For both enzymes, we will collect crystal structures of optimal chimeras.

[0185] Exemplary photoswitch construct sequences for use in expressing in mammalian cells or within an operon for microbial cells. In some embodiments, the sequences may be optimized for microbial expression.

TABLE-US-00001 PPTP1B-LOV2,version7.1(T406A):DNAsequenceSEQIDNO:12: ATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAGTCCGGGAGCTGGGCGGCC ATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCAAGCT TCCTAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTC GGATTAAACTACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGG AAGAAGCCCAAAGGAGTTACATTCTTACCCAGGGCCCTTTGCCTAACACATGCGGT CACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGGGTGTCGTCATGCTCAACA GAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGAAGAA AAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATC AAGTCATATTATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAAC TCGAGAGATCTTACATTTCCACTATACCACATGGCCTGACTTTGGAGTCCCTGAAT CACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGC CCGGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTGGA ACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTC TTCCGTTGATATCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGA TCCAGACAGCCGACCAGCTGCGCTTCTCCTACCTGGCTGTGATCGAAGGTGCCAA ATTCATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAGGAGCTTTCCCACGAG GACGCTGCTACACTTGAACGTATTGAGAAGAACTTTGTCATTACTGACCCAAGGTT GCCAGATAATCCCATTATATTCGCGTCCGATAGTTTCTTGCAGTTGACAGAATATAG CCGTGAAGAAATTTTGGGAAGAAACTGCAGGTTTCTACAAGGTCCTGAAACTGATC GCGCGACAGTGAGAAAAATTAGAGATGCCATAGATAACCAAACAGAGGTCACTGTT CAGCTGATTAATTATACAAAGAGTGGTAAAAAGTTCTGGAACCTCTTTCACTTGCAG CCTATGCGAGATCAGAAGGGAGATGTCCAGTACTTTATTGGGGTTCAGTTGGATG GAACTGAGCATGTCCGAGATGCTGCCGAGAGAGAGGGAGTCATGCTGATTAAGAA AACTGCAGAAAATATTGATGAGGCGGCAAAAGAACTTCTCGAGCACCACCACCAC CACCACTGA Proteinsequence:SEQIDNO:13: MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKL HQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEK GSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHY TTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMD KRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELS HEDAATLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATV RKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVR DAAEREGVMLIKKTAENIDEAAKELLEHHHHHH PTP1B-LOV2,version7.1(S286A):DNAsequence:SEQIDNO:14: ATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAGTCCGGGAGCTGGGCGGCCAT TTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCAAGCTTCC TAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTCGGA TTAAACTACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGGAA GAAGCCCAAAGGAGTTACATTCTTACCCAGGGCCCTTTGCCTAACACATGCGGTCAC TTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGGGTGTCGTCATGCTCAACAGAGT GATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGAAGAAAAA GAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATCAAG TCATATTATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAACTCG AGAGATCTTACATTTCCACTATACCACATGGCCTGACTTTGGAGTCCCTGAATCACC AGCCTCATTCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGCCCGGA GCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTGGAACCTTCTG TCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGA TATCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAG CCGACCAGCTGCGCTTCTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGG GGGACTCTGCCGTGCAGGATCAGTGGAAGGAGCTTTCCCACGAGGACGCTACTACA CTTGAACGTATTGAGAAGAACTTTGTCATTACTGACCCAAGGTTGCCAGATAATCCC ATTATATTCGCGTCCGATAGTTTCTTGCAGTTGACAGAATATAGCCGTGAAGAAATT TTGGGAAGAAACTGCAGGTTTCTACAAGGTCCTGAAACTGATCGCGCGACAGTGAG AAAAATTAGAGATGCCATAGATAACCAAACAGAGGTCACTGTTCAGCTGATTAATT ATACAAAGAGTGGTAAAAAGTTCTGGAACCTCTTTCACTTGCAGCCTATGCGAGATC AGAAGGGAGATGTCCAGTACTTTATTGGGGTTCAGTTGGATGGAACTGAGCATGTCC GAGATGCTGCCGAGAGAGAGGGAGTCATGCTGATTAAGAAAACTGCAGAAAATATT GATGAGGCGGCAAAAGAACTTCTCGAGCACCACCACCACCACCACTGA Proteinsequence:SEQIDNO:15: MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKL HQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEK GSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHY TTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMD KRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSAVQDQWKELS HEDATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATV RKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVR DAAEREGVMLIKKTAENIDEAAKELLEHHHHHH TCPTP-LOV2,bestversion: DNAsequence:SEQIDNO:16: ATGCCCACCACCATCGAGCGGGAGTTCGAAGAGTTGGATACTCAGCGTCGCTGGCA GCCGCTGTACTTGGAAATTCGAAATGAGTCCCATGACTATCCTCATAGAGTGGCCAA GTTTCCAGAAAACAGAAATCGAAACAGATACAGAGATGTAAGCCCATATGATCACA GTCGTGTTAAACTGCAAAATGCTGAGAATGATTATATTAATGCCAGTTTAGTTGACA TAGAAGAGGCACAAAGGAGTTACATCTTAACACAGGGTCCACTTCCTAACACATGC TGCCATTTCTGGCTTATGGTTTGGCAGCAGAAGACCAAAGCAGTTGTCATGCTGAAC CGCGTGATGGAGAAAGGTTCGTTAAAATGTGCACAGTACTGGCCAACAGATGACCA AGAGATGCTGTTTAAAGAAACAGGATTCAGTGTGAAGCTCTTGTCAGAAGATGTGA AGTCGTATTATACAGTACATCTACTACAATTAGAAAATATCAATAGTGGTGAAACCA GAACAATATCTCACTTTCATTATACTACCTGGCCAGATTTTGGAGTCCCTGAATCACC AGCTTCATTTCTCAATTTCTTGTTTAAAGTGAGAGAATCTGGCTCCTTGAACCCTGAC CATGGGCCTGCGGTGATCCACTGTAGTGCAGGCATTGGGCGCTCTGGCACCTTCTCT CTGGTAGACACTTGTCTTTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTG ATATCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAG ACAGCCGACCAGCTGCGCTTCTCCTACCTGGCTGTGATCGAAGGTGCCAAATT CATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAGGAGCTTTCCCACGAGG ACGCTGCTACACTTGAACGTATTGAGAAGAACTTTGTCATTACTGACCCAAGGTTGC CAGATAATCCCATTATATTCGCGTCCGATAGTTTCTTGCAGTTGACAGAATATAGCC GTGAAGAAATTTTGGGAAGAAACTGCAGGTTTCTACAAGGTCCTGAAACTGATCGC GCGACAGTGAGAAAAATTAGAGATGCCATAGATAACCAAACAGAGGTCACTGTTCA GCTGATTAATTATACAAAGAGTGGTAAAAAGTTCTGGAACCTCTTTCACTTGCAGCC TATGCGAGATCAGAAGGGAGATGTCCAGTACTTTATTGGGGTTCAGTTGGATGGAAC TGAGCATGTCCGAGATGCTGCCGAGAGAGAGGGAGTCATGCTGATTAAGAAAACTG CAGAAAATATTGATGAGGCGGCAAAAGAACTTCTCGAGCACCACCACCACCACCA CTGA TheunderlinedlettersindicatesequencefromPTPIB.Protein sequence:SEQIDNO:17: MPTTIEREFEELDTQRRWQPLYLEIRNESHDYPHRVAKFPENRNRNRYRDVSPYDHSRV KLQNAENDYINASLVDIEEAQRSYILTQGPLPNTCCHFWLMVWQQKTKAVVMLNRVM EKGSLKCAQYWPTDDQEMLFKETGFSVKLLSEDVKSYYTVHLLQLENINSGETRTISHF HYTTWPDFGVPESPASFLNFLFKVRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLLL MDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWK ELSHEDAATLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDR ATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTE HVRDAAEREGVMLIKKTAENIDEAAKELLEHHHHHH TCPTP-LOV2V2:DNAsequence:SEQIDNO:18: ATGCCCACCACCATCGAGCGGGAGTTCGAAGAGTTGGATACTCAGCGTCGCTGGCA GCCGCTGTACTTGGAAATTCGAAATGAGTCCCATGACTATCCTCATAGAGTGGCCAA GTTTCCAGAAAACAGAAATCGAAACAGATACAGAGATGTAAGCCCATATGATCACA GTCGTGTTAAACTGCAAAATGCTGAGAATGATTATATTAATGCCAGTTTAGTTGACA TAGAAGAGGCACAAAGGAGTTACATCTTAACACAGGGTCCACTTCCTAACACATGC TGCCATTTCTGGCTTATGGTTTGGCAGCAGAAGACCAAAGCAGTTGTCATGCTGAAC CGCATTGTGGAGAAAGAATCGGTTAAATGTGCACAGTACTGGCCAACAGATGACCA AGAGATGCTGTTTAAAGAAACAGGATTCAGTGTGAAGCTCTTGTCAGAAGATGTGA AGTCGTATTATACAGTACATCTACTACAATTAGAAAATATCAATAGTGGTGAAACCA GAACAATATCTCACTTTCATTATACTACCTGGCCAGATTTTGGAGTCCCTGAATCACC AGCTTCATTTCTCAATTTCTTGTTTAAAGTGAGAGAATCTGGCTCCTTGAACCCTGAC CATGGGCCTGCGGTGATCCACTGTAGTGCAGGCATTGGGCGCTCTGGCACCTTCTCT CTGGTAGACACTTGTCTTTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGAT ATCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGC CGACCAGCTGCGCTTCTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGG GGACTCTTCCGTGCAGGATCAGTGGAAGGAGCTTTCCCACGAGGACGCTGCTACACT TGAACGTATTGAGAAGAACTTTGTCATTACTGACCCAAGGTTGCCAGATAATCCCAT TATATTCGCGTCCGATAGTTTCTTGCAGTTGACAGAATATAGCCGTGAAGAAATTTT GGGAAGAAACTGCAGGTTTCTACAAGGTCCTGAAACTGATCGCGCGACAGTGAGAA AAATTAGAGATGCCATAGATAACCAAACAGAGGTCACTGTTCAGCTGATTAATTATA CAAAGAGTGGTAAAAAGTTCTGGAACCTCTTTCACTTGCAGCCTATGCGAGATCAGA AGGGAGATGTCCAGTACTTTATTGGGGTTCAGTTGGATGGAACTGAGCATGTCCGAG ATGCTGCCGAGAGAGAGGGAGTCATGCTGATTAAGAAAACTGCAGAAAATATTGAT GAGGCGGCAAAAGAACTTCTCGAGCACCACCACCACCACCACTGA Proteinsequence:SEQIDNO:19: MPTTIEREFEELDTQRRWQPLYLEIRNESHDYPHRVAKFPENRNRNRYRDVSPYDHSRV KLQNAENDYINASLVDIEEAQRSYILTQGPLPNTCCHFWLMVWQQKTKAVVMLNRIVE KESVKCAQYWPTDDQEMLFKETGFSVKLLSEDVKSYYTVHLLQLENINSGETRTISHFH YTTWPDFGVPESPASFLNFLFKVRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLLLM DKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKEL SHEDAATLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRAT VRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHV RDAAEREGVMLIKKTAENIDEAAKELLEHHHHHH

[0186] FRET sensors. Forster resonance energy transfer (FRET) is contemplated for use to monitor the activity of PTP1B in living cells. Sensor exhibits a 20% reduction in FRET signal when treated with Src kinase (FIG. 21B). Previous imaging studies indicate that a 20% change in FRET is sufficient to monitor intracellular kinase activity.sup.54-56. To enhance spatial resolution in imaging studies, we will attempt to optimize our sensor further (and use it to measure the activity of PTP1B in vitro).

[0187] Exemplary FRET sensors: underlined mClover3-SH2-Linker-Bold Substrate-underlined and Bold mRuby3.

TABLE-US-00002 mClover3-mRuby3:DNAsequence:SEQIDNO:20: ATGCATCATCATCATCATCATGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGC GGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGT GGCCTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGC CATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCAAGGACGACGGTACCT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTA CAACTTCAACAGCCACTACGTCTATATCACGGCCGACAAGCAGAAGAACTGCATCA AGGCTAACTTCAAGATCCGCCACAACGTTGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCCATCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACA TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATTACACATGGCATGGACGAGCTGT ACAAGTGGTATTTTGGGAAGATCACTCGTCGGGAGTCCGAGCGGCTGCTGCTCAACC CCGAAAACCCCCGGGGAACCTTCTTGGTCCGGGAGAGCGAGACGACAAAAGGTGCC TATTGCCTCTCCGTTTCTGACTTTGACAACGCCAAGGGGCTCAATGTGAAGCACTAC AAGATCCGCAAGCTGGACAGCGGCGGCTTCTACATCACCTCACGCACACAGTTCAG CAGCCTGCAGCAGCTGGTGGCCTACTACTCCAAACATGCTGATGGCTTGTGCCACCG CCTGACTAACGTCTGTGGGTCTACATCTGGATCTGGGAAGCCGGGTTCTGGTGAGGG TTCTTGGATGGAGGACTATGACTACGTCCACCTACAGGGGGAGCTCGTGTCTAA GGGCGAAGAGCTGATCAAGGAAAATATGCGTATGAAGGTGGTCATGGAAGGTT CGGTCAACGGCCACCAATTCAAATGCACAGGTGAAGGAGAAGGCAGACCGTAC GAGGGAACTCAAACCATGAGGATCAAAGTCATCGAGGGAGGACCCCTGCCATT TGCCTTTGACATTCTTGCCACGTCGTTCATGTATGGCAGCCGTACTTTTATCAA GTACCCGGCCGACATCCCTGATTTCTTTAAACAGTCCTTTCCTGAGGGTTTTAC TTGGGAAAGAGTTACGAGATACGAAGATGGTGGAGTCGTCACCGTCACGCAGG ACACCAGCCTTGAGGATGGCGAGCTCGTCTACAACGTCAAGGTCAGAGGGGTA AACTTTCCCTCCAATGGTCCCGTGATGCAGAAGAAGACCAAGGGTTGGGAGCC TAATACAGAGATGATGTATCCAGCAGATGGTGGTCTGAGAGGATACACTGACA TCGCACTGAAAGTTGATGGTGGTGGCCATCTGCACTGCAACTTCGTGACAACTT ACAGGTCAAAAAAGACCGTCGGGAACATCAAGATGCCCGGTGTCCATGCCGTT GATCACCGCCTGGAAAGGATCGAGGAGAGTGACAATGAAACCTACGTAGTGCA ACGCGAAGTGGCAGTTGCCAAATACAGCAACCTTGGTGGTGGCATGGACGAGC TGTACAAGTAA Proteinsequence:SEQIDNO:21: MHHHHHHVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTG KLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTR AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKANFKIRH NVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLEFVTAA GITHGMDELYKWYFGKITRRESERLLLNPENPRGTFLVRESETTKGAYCLSVSDFDNAK GLNVKHYKIRKLDSGGFYITSRTQFSSLQQLVAYYSKHADGLCHRLTNVCGSTSGSGKP GSGEGSWMEDYDYVHLQGELVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGE GRPYEGTQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFT WERVTRYEDGGVVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEP NTEMMYPADGGLRGYTDIALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVD HRLERIEESDNETYVVQREVAVAKYSNLGGGMDELYK

[0188] Exemplary Mammalian expression vector(s) for expressing a photoswitch construct in a mammalian cell.

[0189] For insertion into a mammalian expression vector, e.g. lentiviral vector, pAcGFP1-C1 (Clontech); PTP1B-LOV2 (above), a promoter, e.g. CMV: SEQ ID NO: 22: GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAG TGAACCGTCAGATC; a RBS, e.g. Kozak consensus translation initiation site: GCCACCATG; an Intergenic spacer (, e.g. P2A: DNA sequence: SEQ ID NO: 23: GGCAGCGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAA CCCCGGCCCC; a protein sequence: SEQ ID NO: 24: GSGATNFSLLKQAGDVEENPGP, etc.

[0190] An exemplary FRET Sensor included: a Promoter: Same as above; a RBS: Same as above, etc.

[0191] Exemplary FRET sensors are contemplated to avoid overlap between the excitation/emission wavelengths of LOV2 (455/495, we note that LOV2 is only weakly fluorescent.sup.70) and our FRET pair (505/515 for Clover and 560/605 for mRuby2), while we still expect to see some crosstalk during imaging, previous three-color imaging studies.sup.71 suggest that it will not interfere with our ability to carry out the experiments described in this section.

Contemplative Embodiments Include but at not Limited to Invadopodia Formation and EGFR Regulation.

[0192] A photoswitchable variant of PTP1B is contemplated to determine if cytosolic PTP1B, released from the ER by proteolysis, is exclusively responsible for regulating the formation of invadopodia, or if ER-bound PTP1B can function similarly. Cancer cell invasion and metastasis is facilitated by the formation of invadopodia, actin-rich protrusions that enable matrix degradation.sup.45.

[0193] Both PTP1B and PTK6 regulate epidermal growth factor receptor (EGFR), a regulator of cell proliferation and migration that exhibits aberrant activity in numerous cancers and inflammatory diseases.sup.51,76. We will use a variant of PTP1B stimulated by red light and a variant of PTK6 stimulated by blue light (or vice versa) to carry out a combinatorial analysis of the cooperative contribution of PTP1B and PTK6 to EGFR regulation within different regions of the cell.

References for Sections I, II, and V are Listed Below and Herein Incorporated by Reference

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III. Genetically Encoded System for Constructing and Detecting Biologically Active Agents: Microbial Inhibitor Screening Systems

[0270] Several types of operons were developed as described herein, each for a specific purpose, including but not limited to testing small molecules for their ability to inhibit, activate, or otherwise modulate a chosen PTP and/or PTK; operons for testing intracellularly provided small molecules for inhibiting, activating, or modulating effects on a chosen PTP and/or PTK; and evolving one or more proteins or small molecules of interest. More specifically, genetic operons were contemplated for insertion, using transfection and breeding techniques well known in the art, for providing microbial cells wherein the activity of an enzyme of interest (e.g., protein tyrosine phosphatase 1, a drug target for the treatment of diabetes, obesity, and cancer) is linked to (i) cellular luminescence, (ii) cellular fluorescence, or (iii) cellular growth. In some embodiments, such operons are modified for use in detecting and/or evolving biologically active metabolites. When modified and/or induced to build various metabolites, the cell will be used for detection of metabolites that inhibit/activate a protein of interest (e.g., PTP1B).

[0271] These operons allow operon-containing microbial cells to be used to carry out the following tasks: Detecting biologically active molecules and non-native biologically active metabolites. When grown in the presence of biologically active molecules as a small molecule that is both (i) cell permeable and (ii) capable of inhibiting a protein of interest (e.g., PTP1B), the cell will enable detection of that molecule. That is, it will exhibit a concentration-dependent response in luminescence, fluorescence, or growth. Many non-native biologically active metabolites have useful pharmaceutical properties. Examples include paclitaxel and artemisinin, plant-derived terpenoids that are used to treat cancer and malaria, respectively. When the metabolic pathways responsible for making such natural metabolites are installed into microbial cells that also contain our operon, those cells will enable detection of interesting metabolite-based biological activities (e.g., the ability to inhibit PTP1B).

[0272] Genetic operons that, when installed into microbial cells, link the activity of an enzyme of interest (e.g., protein tyrosine phosphatase 1B, a drug target for the treatment of diabetes, obesity, and cancer) to (i) cellular luminescence, (ii) cellular fluorescence, or (iii) cellular growth.

[0273] Detect and/or evolve biologically active metabolites. When modified and/or induced to build various metabolites, the cell will enable detection of metabolites that inhibit/activate a protein of interest (e.g., PTP1B).

[0274] These operons allow operon-containing microbial cells to be used to carry out the following tasks: Detecting biologically active molecules and non-native biologically active metabolites. When grown in the presence of a biologically active molecules as a small molecule that is both (i) cell permeable and (ii) capable of inhibiting a protein of interest (e.g., PTP1B), the cell will enable detection of that molecule. That is, it will exhibit a concentration-dependent response in luminescence, fluorescence, or growth. Many non-native biologically active metabolites have useful pharmaceutical properties. Examples include paclitaxel and artemisinin, plant-derived terpenoids that are used to treat cancer and malaria, respectively. When the metabolic pathways responsible for making such natural metabolites are installed into microbial cells that also contain our operon, those cells will enable detection of metabolite-based biological activities (e.g., the ability to inhibit PTP1B).

[0275] In some embodiments, methods of evolving molecules may be modified from Moses, et al., Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Phytologist, Volume 200, Issue 1, where this reference describes synthesis of artemisinic acid, the precursor of the antimalarial drug artemisinin, as diterpenoids expressed in E. coli. Further, enzyme engineering or directed evolution of terpenoid biosynthetic enzymes, e.g. engineer enzymes to accept unnatural substrates and to catalyze regions and stereospecific reactions with an efficiency comparable with that of the natural enzymes is described, along with discussions on enhancing the production of terpenoids in Escherichia coli. In some embodiments, methods of evolving molecules may be modified from Badran, et al., Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature, Vol 533:58, 2016, where this reference describes a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac.

A. Protein Evolving Systems and Evolving Biologically Active Metabolites.

[0276] In some embodiments, methods of evolving molecules may be used to construct drug leads that can be readily synthesized in microbial hosts. It addresses a longstanding challenge the development of low-cost pharmaceuticalsby using a sophisticated set of biophysical tools and analytical methodologies to narrow the molecular search space in lead discovery, and by explicitly considering the biosynthetic accessibility of therapeutic molecules. The approach, which departs from contemporary efforts to use microbial systems for the synthesis of clinically approved drugs and their precursors, is unique in its focus on using biology for the systematic construction of new molecules. It will accelerate the rateand lower the costof pharmaceutical development.

[0277] The development of a drug requires optimization of many of its pharmacological propertiesaffinity, absorption, distribution, metabolism, excretion, toxicology, pharmacokinetics, and pharmacodynamicsi. The first of these propertiesprotein-ligand binding affinitygenerally determines whether the others are worth measuring or enhancing, and, thus, represents a property of drug leads.sup.2. Despite advances in computational chemistry and structural biology, the rational design of ligands that bind tightly to proteinsligands, henceforth, referred to collectively as inhibitorsremains exceptionally difficult.sup.3; as a result, the development of drugs often begins with screens of large libraries of molecules.sup.4. An inhibitor, once discovered, must be synthesized in quantities sufficient for subsequent analysis, optimization, formulation, and clinical evaluation.

[0278] The difficulties associated with developing protein inhibitors are particularly problematic for natural products. These molecules, which account for over 50% of clinically approved drugs, tend to have favorable pharmacological properties (e.g., membrane permeability).sup.5. Unfortunately, their low natural titerswhich hamper the extraction of testable quantities from natural sourcesand their chemical complexitywhich complicates chemical synthesismake the preparation of quantities sufficient for post-screen analyses time-consuming and expensive.sup.6.

[0279] In some embodiments, enzymes are contemplated for use to construct terpenoid inhibitors that can be synthesized in ESCHERICHIA COLI; such an approach takes advantage of the chemical diversity (and generally favorable pharmacological properties) of natural products without the constraints of their natural scarcity. In some embodiments, detailed biophysical study of the molecular-level origin and thermodynamic basis of affinity and activity in protein-terpenoid interactions are included for the rapid construction of high-affinity inhibitors. In some embodiments, development of selective inhibitors of protein tyrosine phosphatase 1B (PTP1B), a target for the treatment of diabetes, obesity, and cancer is contemplated in part for using enzymes to evolve readily synthesizable drug leads.

Structurally Varied Terpenoids with Different Affinities for the Allosteric Binding Pocket of Protein Tyrosine Phosphatase 1B (PTP1B).

[0280] Hypothesis. Results indicate that abietic acid, a mono-carboxylated variant of abietadiene, is an allosteric inhibitor of PTP1B. Derivatives or structural analogs of abietadiene that differ in stereochemistry, shape, size, and/or chemical functionality (including carboxylation position) are likely to have different affinities for the allosteric binding pocket of PTP1B.

[0281] In some embodiments, (i) mutants of abietadiene synthase, cytochrome P450s, and halogenases are contemplated for use to make structural variants of abietadiene, (ii) GC/MS to identify those variants, (iii) preparative HPLC and flash chromatography to isolate them, and (iii) isothermal titration calorimetry to determine their free energies, enthalpies, and entropies of binding. In some embodiments, a set of structurally varied inhibitors with (i) affinities that differ by 100-fold and/or (ii) enthalpies and entropies of binding that suggest alternative binding geometries is contemplated.

To Examine the Molecular Basis and Thermodynamic Origin of Affinity and Activity in Enzyme-Terpenoid Interactions.

[0282] Hypothesis. Enzymes that bind, functionalize, and/or synthesize terpenoids possess large nonpolar binding pockets. We hypothesize that both (i) the affinity of an enzyme for terpenoids and (ii) the activity of an enzyme ON terpenoids is determined by the general shape and hydration structure of its binding pocket, not the position of specific protein-terpenoid contacts.

[0283] In some embodiments, a sophisticated set of biophysical tools (isothermal titration calorimetry, X-ray crystallography, molecular dynamics (MD) simulations, and NMR spectroscopy) are contemplated for use to (i) determine how protein-ligand contacts, rearrangements of water, and conformational constraints contribute to differences in affinity between terpenoid inhibitors and to (ii) develop a set of empirical relationships that predict how mutations in terpene synthases and terpene-functionalizing enzymes influence general attributes (e.g., shape) of their products.

To Evolve High-Affinity Terpenoid Inhibitors of PTP1B.

[0284] Hypothesis. Mutants from secondary metabolism (e.g., terpene synthases, cytochrome P450s, and halogenases) are highly promiscuous; a single mutation in or near their active sites can dramatically alter their product profiles. Mutagenesis of a small number (i.e., 2-4) of such enzymes, selected for their ability to synthesize and/or functionalize diterpenoids, will enable the development of inhibitors of PTP1B with sub-micromolar affinities.

[0285] In some embodiments, high-affinity inhibitors of PTP1B by pairing (i) high-throughput methods for detecting inhibitors with (ii) site-saturation and random mutagenesis is contemplated. For (i) we will develop four alternative fluorescence or growth-coupled assays to screen libraries of mutated pathways (and their respective products). For (ii) we use biostructural analyses and sequence alignments to identify residues likely to yield enzymes with favorable product profiles.

To Identify Structure-Activity Relationships that Enable the Evolution of Terpenoid Inhibitors of Arbitrary Protein Targets.

[0286] Hypothesis. Proteins that interact with similar classes of molecules (through binding or catalysis) have structurally similar binding pockets. Methods for evaluating these structural similaritiesand their implications for enzyme activitymay enable the identification of enzymes capable of synthesizing inhibitors of ANY specified protein.

[0287] In some embodiments, a biophysical framework for using the crystal structure of a protein as a starting point to identify enzymes capable of synthesizing inhibitors of that protein is contemplated. We will examine (and formalize) structural relationships between (i) the active sites of enzymes used to synthesize allosteric inhibitors of PTP1B and (ii) the allosteric binding pocket of PTP1B, and we will validate these relationships by using them to identifyand, then, test-new enzymes capable of synthesizing inhibitors of PTP1B and (separately) undecaprenyl diphosphate synthase, a target for the treatment of antibiotic-resistant bacterial infections.

Diabetes, Obesity, and Cancer.

[0288] Protein tyrosine phosphatase 1B (PTP1B) contributes to insulin resistance in type 2 diabetes.sup.7, leptin resistance in obesity.sup.8, and tumor growth in breast, colorectal, and lung cancers.sup.9,11. To date, the development of selective, tight-binding inhibitors of PTP1B (i.e., treatments for diabetes, obesity, and cancer) has been hindered by the structure of its active site, where polar residues limit tight binding to charged, membrane-impermeable molecules, and where structural similarities to the active sites of other protein tyrosine phosphatases (PTPs) lead to off-target interactions.sup.12,14. In this proposal, we will construct selective inhibitors of PTP1B that bind to its C-terminal allosteric site, a largely nonpolar region that is not conserved across phosphatases. Previous screens of large molecular libraries have identified several ligands that bind to this site, but have yet to yield clinically approved drugs.sup.6,13. The identification of new molecular alternativesa feat tackled in this proposal-remains a goal in efforts to develop selective PTP1 B-inhibiting therapeutics.

[0289] Development of pharmaceuticals. The development of enzyme inhibitorsor leads represents an expensive part of drug development; for each successful drug, lead identification and optimization takes an average of 3 years and $250M to complete (?20-30% of the total time and cost to bring a drug to market).sup.17. By narrowing the molecular search space in lead discovery, by enabling rapid construction of structurally-varied leads (often referred to as backups.sup.18), and by facilitating scale-up of molecular synthesis, the technology developed in this proposal could accelerate the rateand lower the costof pharmaceutical development.

[0290] Molecular recognition. The hydrophobic effectthe free energetically favorable association of nonpolar species in aqueous solutionis, on average, responsible for ?75% of the free energy of protein-ligand association.sup.19. Unfortunately, hydrophobic interactions between ligands and proteins-which differ dramatically in rigidity, topography, chemical functionality, and hydration structure-remain difficult to predict.sup.20. This study uses detailed biophysical analyses and explicit-water calculations to examine the thermodynamic basis of hydrophobic interactions between terpenoids and protein binding pockets. It will develop a model systemand corresponding conceptual frameworkfor studying the hydrophobic effect in the context of structurally varied protein-ligand complexes, for accounting for that effect in the design of biosynthetic pathways, and for exploiting it in the construction of new drug leads.

Biosynthesis of New Natural Products.

[0291] Synthetic biology offers a promising route to the discovery and production of natural products. When the metabolic machinery of one organism is installed into a genetically tractable production host (e.g., S. CEREVISIAE or E. COLI), it enables the synthesis of complex compounds at high titers (relative to the native host). This approach has enabled the efficient production of pharmaceutically relevant metabolites from unculturable or low-yielding organisms.sup.21,22, but, unfortunately, requires large investments of time and resources in pathway discovery and optimization; its use, as a result, is generally limited to the low-throughput characterization of newly discovered gene clusters or to the production of known, pharmaceutically relevant molecules (e.g., paclitaxel, artemisinin, or opioids).sup.22,24.

[0292] In some embodiments, a strategy for using synthetic biology to build new molecular function is contemplated. It begins with a pathologically relevant protein target and engineers pathway enzymes to produce molecules that selectively inhibit that target. This approach will yield molecules that can be produced in microbial hosts without extensive pathway optimization (it relies on enzymes that are expressible by default); it will, thus, expand the use of synthetic biology to the production of leads and backups. It is not a replacement for conventional approaches to the synthesis of complex natural products, but rather, a complementary strategy for constructing new compounds that will enhance the efficiency with which pharmaceuticals are developed.

[0293] In the presence of mutated metabolic pathways (e.g., version of a plant-based terpenoid-producing pathway in which the terpene synthase has been mutated), our operon will enable screens of large numbers of metabolites for their ability to inhibit our protein of interest (e.g., PTP1B). Such a platform could be used to evolve metabolites with specific biological activities.

[0294] Detect and/or evolve highly selective molecules. We have developed an idea for a version of our operon to detect molecules that inhibit one protein over a highly similar protein. Screens for molecular selectivity are, at present, remain very difficult.

[0295] Advantages of methods and systems described herein, over some other systems for detecting small molecule inhibitors includes but is not limited to enabling the detection of molecules that modulate or change the catalytic activity of an enzyme. Moreover, some embodiments of the systems described herein allow for the detection of test molecules that change the activity of an enzyme by binding anywhere on its surface. As one example, detection of an inhibitor is contemplated that inactivates PTP1B by binding to its C-terminal allosteric site; this binding event, which distorts catalytically essential motions of the WPD loop, would not necessarily prevent enzyme-substrate association. U.S. Pat. No. 6,428,951, herein incorporated by reference in its entirety, in contrast, enables the detection of molecules that prevent enzyme-substrate binding by competing for substrate binding sites (i.e., the active site). As another example, detection of molecules that activate an enzyme of interest is contemplated as an embodiment. U.S. Pat. No. 6,428,951, herein incorporated by reference in its entirety, in contrast, has methods that merely detect molecules that prevent enzymes from binding to their substrates, or that otherwise change the affinity of enzymes for their substrates. As another example, detection of molecules that do not require an enzyme and substrate to interact with any particular affinity, orientation, or half-life is contemplated as an embodiment. U.S. Pat. No. 6,428,951, herein incorporated by reference in its entirety, in contrast, requires an enzyme and substrate to bind one another with an affinity and orientation that enable assembly of a split reporter. As a result, it may require modifications to the enzyme; in contrast, the inventors use a substrate trapping mutant of PTP1B to improve its affinity for a substrate domain.

[0296] As another example, some embodiments enables the detection of inhibitors of wild-type enzymes. Tu S., U.S. Pat. No. 6,428,951, herein incorporated by reference in its entirety, in contrast, requires enzymes to be fused to one-half of a split reporter.

[0297] Further, the following two publications are examples of methods that for detecting molecules that merely disrupt the binding of an enzyme to a substrate. This characteristic, among others, is in contrast to U.S. Pat. No. 6,428,951. Protein fragment complementation assays for the detection of biological or drug interactions. Pub. Date: Jan. 31, 2008, herein incorporated by reference in its entirety, which describes a high throughput bacteria based protein-fragment complementation assays (PCAs) wherein when two protein fragments derived from the enzyme dihydrofolate reductase (DHFR), coexpressed as fusion molecules in Escherichia coli, that interact in the absence of an inhibitor, then concentration dependent colony growth was observed. This reference states that PCA can be adapted to detecting interactions of proteins small molecules and provide examples, including complementary fragment fusions and a bait-fused fragment. In fact, protein tyrosine phosphatase PTP1B was provided in an example for detecting enzyme substrate interactions and an example of survival assay for detecting protein substrate interactions using aminoglycoside kinase (AK), an example of antibiotic resistance marker used for dominant selection of an E. coli,-based PCA. Further, a PCA is described as being applied to identify small molecule inhibitors of enzymes; natural products or small molecules from compound libraries of potential therapeutic value; may be used as survival assay for library screening; for detecting endogenous DHFR inhibitors, e.g. rapamycin; and for protein-drug interactions. Expression of PCA complementary fragments and fused cDNA libraries/target genes can be assembled on single plasmids as individual operons under the control of separate inducible or constitutive promoters with interceding region sequences, e.g. derived from a mel operon, or have polycistronic expression. The PCA can be adapted to detecting interactions of proteins with small molecules. In this conception, two proteins are fused to PCA complementary fragments, but the two proteins do not interact with each other. The interaction must be triggered by a third entity, which can be any molecule that will simultaneously bind to the two proteins or induce an interaction between the two proteins by causing a conformational change in one or both of the partners. Moreover, exemplary applications of the PCA Strategy in bacteria to protein engineering/evolution to generate peptides or proteins with novel binding properties that may have therapeutic value using phage display technology. One example of evolution produced novel zipper sequences; other examples of evolutions were described to produce endogenous toxins.

[0298] WO2004048549. Dep-1 Receptor Protein Tyrosine Phosphatase Interacting Proteins And Related Methods. Published Jun. 10, 2004, herein incorporated by reference in its entirety; describes screening assays for inhibitors that alter the interaction between a PTP and a tyrosine phosphorylated protein that is a substrate of the PTP, e.g. dephosphorylation by Density Enhanced Phosphatase-1 (DEP-1) of a DEP-1 substrate. DEP-1 polypeptides can be expressed in bacteria cells, including E. coli, under the control of appropriate promoters, e.g. E. coli arabinose operon (P.sub.BAD or P.sub.ARA). This reference is similarly limited in focus as U.S. Pat. No. 6,428,951, herein incorporated by reference in its entirety; it enables the detection of molecules that disrupt the binding of a substrate to an enzyme, rather than the detection of molecules that modulate (i.e., enhance or reduce) the activity of an enzyme.

[0299] Advantages of methods and systems described herein, over some other systems for detecting small molecule inhibitors includes but is not limited to enabling the evolution of metabolites that change the catalytic activity of an enzyme. The technology described in Badran, et al., Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature, Vol 533:58, 2016, herein incorporated by reference in its entirety; and the platform of continuous evolution in general, has been used to evolve proteins with different affinities for other proteins/peptide substrates. It has not, however, been used to evolve enzymes that produce small molecules (i.e., metabolites) that alter the activities of enzymes or the strength of protein-protein interactions.

[0300] Another advantage of methods and systems described herein, over some other systems for detecting small molecule inhibitors includes the discovery of metabolites with targeted biological activities but unknown structures (e.g., the ability to inhibit protein tyrosine phosphatase 1i). There are many inventions relevant to the production of terpenoids in E. coli or S. cerevisiae (e.g., Moses, et al., Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Phytologist, Volume 200, Issue 1, where this reference describes synthesis of artemisinic acid, the precursor of the antimalarial drug artemisinin, as diterpenoids expressed in E. coli. Further, enzyme engineering or directed evolution of terpenoid biosynthetic enzymes, e.g. engineer enzymes to accept unnatural substrates and to catalyze regions and stereospecific reactions with an efficiency comparable with that of the natural enzymes is described, along with discussions on enhancing the production of terpenoids in Escherichia coli.); in many cases, the metabolic pathways responsible for making these terpenoids are mutated to improve production levels. However, the use of biosensors (i.e., constructs that report on the concentrations of various metabolites) has focused on the detection of specific intermediates (e.g., farnesyl pyrophosphate, a precursor to terpenoids) not for combining (i) mutagenesis of a metabolic pathway and (ii) a biosensor for specific biological activity (e.g., the ability to inhibit PTP1B) for the discovery of new biologically active molecules (which may possess unknown structures).

High-Throughput Metabolic Engineering.

[0301] Microbial pathways are most efficiently optimized with high-throughput screens. Unfortunately, at present, such screens are sparse, and those available rely on signals (e.g., absorbance or fluorescence, association with a product-specific transcription factor, or growth permitted by an essential metabolite) that are difficult to extend to broad classes of molecules (e.g., those without distinct optical or metabolic properties).sup.27. The proposed work develops high-throughput screens for terpenoids with a targeted activitythe ability to inhibit PTP1B-rather than a targeted structure; these activity-focused screens could be broadly useful for building (i.e., evolving) new biologically active small molecules.

[0302] Identification of new inhibitors: a starting point. We recently discovered that abietic acid, the primary component of resin acid, is an allosteric inhibitor of PTP1B (FIG. 22). We will use abietadiene, the immediate precursor of this molecule, as a starting point for the design of inhibitors. Abietadiene has several attributes that make it particularly compatible with our approach: (i) It can be synthesized in E. COLI at titers (200 mg/L) that permit purification, NMR analysis, and calorimetric studies.sup.28, (ii) Mutants of its associated terpene synthase-abietadiene synthaseyield a range of hydrocarbon scaffolds that differ in stereochemistry, shape, and size (and that still enable analysis and purification).sup.29-31, (iii) Itand similar moleculescan be functionalized by cytochrome P450s and halogenases.sup.32-34.

[0303] Metabolic engineering. We have engineered a strain of E. COLI to produce abietadiene at titers (>150 mg/L) sufficient to permit the analytical methods (i.e., GC/MS, ITC, and NMR). Our biosynthetic pathway has two requisite operons: MBIS, which converts (RJ-mevalonate to farnesyl pyrophosphate (FPP), and TS, which converts farnesyl pyrophosphate to abietadiene. One optional operonMevT, which converts acetyl-CoA to (RJ-mevalonateis necessary when mevalonate is not included in the media.sup.35. The plasmids pMevT and pMBIS were developed by the Keasling Laboratory.sup.36. The plasmid pTS, which contains abietadiene synthase (ABS) from ABIES GRANDIS, was developed as in Morrone.sup.28 with a gene for geranylgeranyl diphosphate synthase from Ajikumar.sup.37.

[0304] Improved inhibitors. We assessed the ability of minor structural perturbations of abietadiene derivatives to yield improved inhibitors by comparing four structurally related (and commercially available) molecules (FIG. 16): abietic, neoabietic, levopimaric, and dihydroabietic acid. Surprisingly, dihydroabietic acid was ten times more inhibitory than abietic acid (K, ?25 uM vs. 250 uM). Our ability to find an improved inhibitor in this small screen suggests that the kinds of structural variations explored in this studyand the sizes of molecular libraries generatedare likely yield improved inhibitors.

[0305] Functionalization of abietadiene. We assessed the ability of mutants of cytochrome P450.sub.bm3 to functionalize abietadiene-like molecules by installing five readily available mutants (G3, KSA-4, 9-1 OA, 139-3, and J, which were engineered for activity on amorphadiene.sup.38 and steroids.sup.39) into our heterologous pathway; three mutants yielded hydroxylated and/or carboxylated products, generating up to 0.3 mg/L of abietic acid (FIG. 17). The abietadiene-functionalizing activity of mutants originally engineered for other targets suggests that we will be able to develop mutants of P450.sub.bm3 with even higher activities on abietadiene-like molecules.

[0306] Biostructural analyses. We have crystallized PTP1B in our lab, collected X-ray diffraction data in collaboration with Peter Zwart at Lawrence Berkeley National Lab (LBNL), and solved its crystal structure (FIG. 17A inset). We have also co-crystallized PTP1B with abietic acid; we will analyze these crystals in late July (first available beam time).

[0307] Recently, we expressed N.sup.15-labeled PTP1B and used it to collect two-dimensional .sup.1H-.sup.15N HSQC spectra in collaboration with Haribabu Arthanari at Harvard Medical School (FIG. 17A main). The spectra include PTP1B bound (separately) to abietic acid and known inhibitors; at present, we are processing the data. Preliminary results (X-ray and NMR) suggest that biostructural studies of PTP1B bound to different inhibitors will be straightforward.

[0308] High-throughput screens. Upon binding to inhibitors (both competitive and allosteric), PTP1B exhibits changes in conformation that quench its tryptophan fluorescence (the basis of one of our four high-throughput screens). FIG. 17B indicates that such quenching can be used to distinguish between inhibitory extract (i.e., a hexane overlay) from an abietadiene-producing strain of E. COLI and non-inhibitory extract from a control strain (i.e., one with a catalytically inactive ABS). FIG. 17C indicates that such changes can also be used to detect 50 uM (15 mg/L) of abietic acid. Our ability to detect (i) abietadiene in culture extract and (ii) abietic acid at low concentrations (i.e., tenfold lower than our titers of abietadiene) suggests that we will be able to detect improved inhibitors of PTP1B, even if they are accompanied by reductions in titer.

[0309] Providing structurally varied terpenoids with different affinities for the allosteric binding pocket. This section describes developing a set of inhibitors with incremental differences in affinity that result from systematic differences in structure. The goal (metric for success): a minimum of ?15 structurally varied inhibitors with (i) affinities for PTP1B that differ by 100-fold and/or (ii) enthalpies and entropies of binding that suggest alternative binding geometries.

[0310] Research plan. In the sections that follow, we use enzymes to build selective terpenoid inhibitors of PTP1B. This enzyme is the initial focus of our work because it is a therapeutic target for diabetes, obesity, and cancer, and it can be expressed, crystallized, and assayed with ease.sup.15. It, thus, serves as a pharmaceutically relevant model system with which to develop a general approach for the enzymatic construction of drug leads.

[0311] Hypothesis for structural changes. In this section, we use promiscuous enzymes to construct terpenoids that differ in stereochemistry, shape, size, and chemical functionality. We believe that these modifications will affect the affinity of ligands for PTP1B by altering (i) their ability to engage in van der Waals interactions with nonpolar residues (e.g., F280, L192, and F196) in the allosteric binding pocket, (ii) their ability to engage in direct or water-mediated hydrogen bonds with proximal polar residues (e.g., N193, E200, and E276), (iii) their ability to engage in halogen bonds with either set of residues, (iv) their influence on molecular conformational constraints, and, (v) their ability to reorganize water during binding. This hypothesis (which is supported, in part, by FIG. 16) motivates the synthetic strategy described herein.

[0312] Stereochemistry, shape, and size. We will begin by using mutants of ABS to generate diterpenoids that differ in stereochemistry and shape FIG. 18A). ABS uses two active sites to catalyze sequential class II (protonation-dependent) and class I (ionization-dependent) cyclization of geranylgeranyl pyrophosphate (GGPP, C.sub.2o) into abietadiene.sup.29. Previous studies indicate that amino acid substitutions in its active sites can alter the stereochemistry or shape of its products.sup.29,31. We will use mutations (new and previously identified) that affect the position of deprotonation, intramolecular protein transfer, or carbocation stability (FIG. 8B). After installing these mutants into E. COLI, we will use GC/MS to search for new products (fragmentation tools such as MetFrag.sup.40 or ACD/MS Fragmenter.sup.41 will facilitate identification of novel compounds).

[0313] We will generate terpenoids that differ in size by using mutations that increase/decrease the volume of the active sites of ABS. Previous attempts to change the substrate specificities of terpene synthases.sup.42,43 suggest that such mutations could enable enhanced activity on farnesyl pyrophosphate (FPP, CI.sub.5) and farnesylgeranyl pyrophosphate (FGPP, C.sub.2s). To synthesize FGPP, we will incorporate an FGPP synthase previously expressed in E. COLI.sup.44.

[0314] We will isolate a subset of new terpenoids with particularly high titers by using flash chromatography and HPLC (a task for which feasibility has been established in several studies.sup.28,31,45), and we will use ITC to measure the free energy (AG.sup.o.sub.birid), enthalpy (AH.sup.o.sub.bind), and entropy (?TAS.sup.o.sub.bind) of binding to PTP1B. Differences in AG.sup.o.sub.bind between ligands will reveal how structural changes affect the strength of binding; differences in AH.sup.o b.sub.ind and ?TAS.sup.o.sub.bi.sub.nd will reveal their influence on binding geometry.sup.46,47.

[0315] Hydroxylation and halogenation. For each of the three ligands selected in 6.1.2, we will use mutants of cytochrome P450 BM3 (P450.sub.bm3) from BACILLUS MEGATERIUM and/or CYP720B4 (P450.sub.72o) from PICEA SITCHENSIS to construct five variants with hydroxyl or carboxyl groups at different positions (FIGS. 19A and 19B). P450.sub.bm3 can hydroxylate a wide range of substrates, including terpenoids.sup.48; P450.sub.72o can carboxylate over 20 diterpenoids, including abietadiene.sup.49. Both enzymes can be expressed in E. COLI.sup.4A.

[0316] We will work with several sets of mutations: For P450.sub.bm3, we will use (i) three (V78A, F87A, and A328L) that permit the stereoselective hydroxylation of sesquiterpenes and diterpenes.sup.50, (ii) five (L75A, M177A, L181A, and L437A) that enable hydroxylation of alkaloids and steroids.sup.51), and (iii) two (F87V and A82F) that permit carboxylation of heteroaromatics (FIG. 18D).sup.52. For P450.sub.7no, we will examine ?10 similar mutations likely to alter the position of oxidation. We will, again, screen each mutant in E. COLI, isolate interesting products, and use ITC to analyze them.

[0317] For each of two high-affinity oxygenated ligands, we will construct six variants with bromide or iodide at different positions (FIG. 18C). These two halogens can engage in halogen bonds with oxygen, nitrogen, or sulfur acceptors in proteins.sup.53, and can bind small nonpolar declivities on their surfaces.sup.54. The energetic contribution associated with both interactions tends to increase from Br to I.sup.54,55 and, thus, lends itself to systematic analysis (i.e., a physical organic approach). To generate halogenated ligands, we will use mutants of tryptophan 6-halogenase (SttH) from STREPTOMYCES TOXYTRICINI and vanadium haloperoxidase (VHPO) from ACARYOCHLORIS MARINA. These enzymes can introduce halogens (chloride, bromide, or iodide) into sp.sup.2-hybridized carbons of alkaloids or terpenoids (before or after cyclization).sup.56,57. For each enzyme, we will examine several mutations known to change regioselectivity (e.g., L460F, P461E, and P452T for SttH.sup.56) and 5-10 mutations likely to change the orientation of bound FIG. 19 Examples: (FIG. 19A) carboxylated, (B) hydroxylated, ligands (FIG. 19E). We will, again, screen (FIG. 19C) and halogenated diterpenoids. (FIG. 19D-E) Residues each mutant in E. COLI and use ITC to targeted for mutagenesis in (FIG. 19D) P450.sub.bm3 and (FIG. 19E) SttH.

IV. Evolving High-Affinity Terpenoid Inhibitors of PTP1B

[0318] This section develops four high-throughput screens for rapidly evaluating the strength of PTP1B inhibitors, and it uses those methods, in conjunction with site-saturation and random mutagenesis, to evolve new inhibitors. The goal: a set of evolved inhibitors with particularly high affinities (K.sub.D {circumflex over ()}1 uM) and/or unpredictable structures (i.e., structures inconsistent with rational design).

[0319] Biological selection. A selection method (i.e., a growth-coupled screen) in which the survival of E. COLI is linked to inhibitor potency will enable rapid screening of extremely large libraries of molecules (10.sup.10).sup.66. In this section, we develop such a method.

[0320] PTP1B catalyzes the dephosphorylationand inactivationof several cell surface receptors. We will use the tyrosine-containing regions of these receptors to build an operon that links inhibition of PTP1B to cell growth. This operon will require six components (FIG. 21A): (i) a substrate domain (the tyrosine-containing region of a receptor) tethered to a DNA-binding protein, (ii) a substrate recognition domain (a protein that binds the tyrosine-containing region after its phosphorylation) tethered to the co subunit of an RNA polymerase, (iii) a tyrosine kinase, (iv) PTP1B, (v) a gene for antibiotic resistance, and (vi) an operator for that gene. With this system, inhibitors of PTP1B will enable binding of the substrate and substrate recognition domains, recruitment of RNA polymerase to the DNA, and transcription of the gene for antibiotic resistance. Previous groups have used similar operons to evolve protein-protein binding partners; here, we take the additional steps of (i) using a protein-protein interaction mediated by enzymes (PTP1B and a kinase) and of (ii) screening that interaction in the presence of potential inhibitors of one of those enzymes.

[0321] We will develop our operon by starting with a luminescence-based system, and we will add an antibiotic resistance gene as a final step. In our preliminary work with a system optimized by Liu et al..sup.67, we obtained a tenfold difference in Lux-based luminescence between a strain expressing two binding partners and a strain expressing one (FIG. 21E; arabinose induces expression of the second partner). We now plan to introduceand test different substrate domains, recognition domains, and kinases (eGFR and Src).

[0322] A FRET sensor for PTP1B activity. A high-throughput screen in which inhibition of PTP1B is linked to cell fluorescence will enable rapid screening via fluorescence-activated cell sorting (FACS). This technique tends to produce more false positives than selection and limits libraries to sizes of 10.sup.7-10.sup.8, but it requires fewer heterologous genes.sup.27,66.

[0323] For this strategy, we will make use of FRET (Forster resonance energy transfer) sensors commonly used to monitor kinase and phosphatase activity in mammalian cells.sup.68,69. These sensors consist of a kinase substrate domain, a short flexible linker, and a phosphorylation recognition domain-all sandwiched between two fluorescent proteins. Phosphorylation of the substrate domain causes it to bind to the recognition domain, inducing FRET between the two fluorescent proteins. In a PTP1B-compatible sensor, inhibitors of PTP1B will increase FRET (FIG. 21). We have begun to develop such a sensor by trying different combinations of substrate domains, recognition domains, and kinases. (Note: FACS enables FRET-based screens.sup.70,71).

[0324] A FRET sensor for changes in the conformation of PTP1B. A FACS-based screen in which changes in cell fluorescence result from binding-induced changes in the conformation of PTP1B would be less generalizable than strategies 2 and 3 (which could be used for any kinase or phosphatase), but would require only one heterologous gene.

[0325] For this strategy, we will make use of a FRET experiment carried out by the Tonks Group.sup.13. These researchers sought to show that the binding of trodusquemine to PTP1B caused the protein to become more compact. To do so, they attached members of a FRET pair to each terminus of the PTP1B (FIG. 21C); upon protein-ligand association, an increase in FRET signal indicated that its termini approached one another. We hypothesize that this construct could be used as a sensor for identifying other molecules that bind to the allosteric site of PTP1B. We will begin by testing it with a variety of known inhibitors (a step the Tonks group did not take).

[0326] Binding-induced changes in the tryptophan fluorescence of PTP1B. A screen in which inhibition of PTP1B is linked to changes in tryptophan fluorescence (FIG. 21D) will enable rapid screening of moderately sized libraries (10.sup.3-10.sup.4).sup.27 in microtiter plates. Our use of binding-induced changes in tryptophan fluorescence is described in 5.6. In future work, we plan to extend this approach to other protein tyrosine phosphatases, many of which are allosteric and possess many tryptophans (e.g., SHP-2, a target for Noonan syndrome.sup.72)

[0327] Mutagenesis. To use our high-throughput screens to evolve inhibitors of PTP1B, we will build libraries of mutated terpenoid pathways by using (i) site-saturation mutagenesis (SSM; we will target binary combinations of sites) and (ii) error-prone PCR (ep-PCR).

[0328] For SSM, we will identify plastic residues likely to accommodate useful mutations by developing functions similar to Eq. 1. This function scores residues based on their ability to accommodate mutations that influence the volume and hydration structure of an active site; S is a metric for the propensity of a residue to permit mutations, cr.sup.2 is the variance in volume of

s=4+RTW(EQ 1}

similarly positioned residues in the active sites of other enzymes, A{circumflex over ()}.sub.W is the variance in hydrophilicity of those residues, and N.sub.V and N.sub.HW are normalization factors. In our preliminary analysis of ABS, we successfully used Eq. 1 (and structure/sequence information from Taxadiene, y-humulene, 5-selenine, and epi-isozizaene synthases) to identify residues for which mutations are known to yield new products (e.g., H348 of ABS).sup.31. We note: Previous attempts to identify plastic residues have scanned each site near the bound substrate.sup.73; our approach will be unique in its inclusion of biophysical considerations from (i) our study of optimal ligand attributes (6.2.1) and (ii) our study of the types of mutations that bring them about (6.2.2). For library construction, we will explore mutating our pathway (i) enzymeby enzyme (e.g., ABS, then P450.sub.bm3, and then VttH) or (ii) at random. The second approach could give us access to structures that might be difficult to find with conventional approaches to lead design.

[0329] To identify structure-activity relationships that enable the evolution of terpenoid inhibitors of arbitrary protein targets. This section develops a biophysical framework for using a crystal structure of a protein to identify enzymes capable of making inhibitors of that protein. The goal: the use of that framework to identifyand, then, test-enzymes capable of synthesizing new inhibitors of PTP1B and (separately) undecaprenyl diphosphate synthase (UPPS), a target for antibiotic-resistant bacterial infections.

[0330] Relationships between binding pockets. We will begin by determining how similarities in specific properties of binding pockets (e.g., volume, polarity, and shape) enable enzymes to synthesize, functionalize, and/or bind similar molecules. This effort will involve comparisons of the allosteric binding pocket of PTP1B with the binding pockets (i.e., active sites) of enzymes involved in inhibitor synthesis. For these comparisons, we will construct two matrices: matrix A in which each element (ay) represents the similarity of a specific property between binding pockets i and j ((0<aij<1, where 1 is highly similar) and matrix B in which each element (by) describes the ability of binding pockets i and j to bind similar molecules (0<by<1, where 1 represents identical binding specificities). The rank of the matrix formed by the product of these two matrices (AB) will suggest the number of independent variables (i.e., active site attributes) necessary to determine the functional compatibility of enzymes in a metabolic pathway; the eigenvalue will suggest the relative importance of the property under study (described by matrix A).

[0331] We will construct matrix A with PyMol- and MD-based analyses of protein crystal structures. We will construct matrix B by examining the binding of functionalized terpenoids and their precursors to each enzyme involved in terpenoid synthesis. Binding affinities for some of these ligand/protein combinations will be measured with ITC; most will be estimated with docking calculations (OEDocking.sup.78).

[0332] The result of this section will be an equation similar to Eq. 2, where J is a metric for an active site's ability to synthesize

J=w.sub.vV+w.sub.pP+WiL+w.sub.wW(Eq. 2)

terpenoids that bind a particular binding pocket; V, P, L, and W represent specific properties of that active site (volume, polarity, longest diameter, and shortest diameter); and w's represent weighting factors. The final number of variables- and their respective weights-will be determined through the above analysis. In parameterizing the equation, we plan to examine different metrics for properties of binding pockets (e.g. shape) and to explore/develop different matrix manipulations.

Validation and Extension.

[0333] The identification of promising active site motifs for inhibitor synthesis will require a search of available protein structural data. We will perform such a search by using PROBIS (probis.nih.gov.sup.79), an alignment-based platform that uses a specified binding site to find similar binding sites on other proteins in the Protein Data Bank. PROBIS can identify similarly shaped binding pockets, even when the protein folds that surround those pockets are different (i.e., it detects similar constellations of amino acids).

[0334] To begin, we will use a PROBIS-based search to identify enzymes with active sites that have some level of structural similarity (we will explore different thresholds) to either (i) the allosteric binding site of PTP1B or (ii) the active sites of enzymes capable of synthesizing inhibitors of PTP1B. Using Eq. 2, we will select enzymes with the most favorable active sites and test them with our platform for inhibitor development).

[0335] We will assess the generalizability of our approach by attempting to construct inhibitors of UPPS, a protein known to bind terpenoids and polycyclic molecules.sup.80. Structure-based searches will use two starting points: (i) UPPS and (ii) mutants of ABS, P450.sub.bm3, or similar enzymes that our biophysical analyses suggest might yield UPPS inhibitors. We will, again, select a subset of enzymes to test with our platform.

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V. Specific Embodiments of Bacterial Systems for Identifying Small Molecules that Modulate the Activity of Enzymes

[0420] As described herein, a strain of Escherichia coli was developed comprising both (i) a genetically encoded system (i.e., a bacterial two-hybrid or B2H system) that links cell survival to the modulation inhibition of a pathologically relevant enzyme from Homo sapiens (i.e., a drug target) and (ii) a pathway for metabolite biosynthesis. The genetically encoded system described herein contains more genetic elements than would traditionally constitute a single operon (e.g. it has more than one promoter), but it is sometimes referred to as an operon.

[0421] More specifically, as described herein, host organisms, e.g. Escherichia (E.) coli, were transformed with up to four plasmids, including a first plasmid (plasmid 1) an expression plasmid comprising a genetically encoded system that links the inhibition of a target enzyme to cell survival, wherein the target enzyme may be chosen for the purpose of identifying molecules that inhibit a specific target enzyme; a second plasmid (plasmid 2) an expression plasmid comprising an operon for expressing at least some of the genes necessary to synthesize products of a metabolic pathway, e.g. a mevalonate-dependent pathway for terpenoid biosynthesis derived from Saccharomyces cerevisiae for providing terpenoid product compounds; a third plasmid (plasmid 3) an expression plasmid comprising at least one additional gene, not present in plasmid (plasmid 2), e.g. a terpene synthase, such as ADS, GHS, ABS, or TXS, for providing desired products, e.g. terpenoid products, such that when the host bacterial expresses plasmids 1 and 2, desired products are not produced until the host bacterial expresses plasmid 3 for completing the pathway for desired compounds; and a fourth plasmid (plasmid 4) comprising additional genetic components specific to the strain of E. coli, e.g., the F-plasmid of S1030 (Addgene 105063).

[0422] Examples of plasmid 1 embodiments are shown in FIG. 33A, 33B, 33D, 33E, FIG. 34, FIG. 35, FIG. 40A, 40B, 40C, 40D, etc.

[0423] In some embodiments, a strain of E. coli used as a host for transformation possesses the ?rpoZ mutation, which enable the system encoded by plasmid 1 to control the expression of a gene for antibiotic resistance.

[0424] In some embodiments, plasmids 2 and/or 3 constitute a pathway for terpenoid biosynthesis. In some embodiments, plasmids 2 and/or 3 constitute a pathway for alkaloid biosynthesis. In some embodiments, plasmids 2 and/or 3 constitute a pathway for polyketide biosynthesis.

[0425] In some embodiments, plasmid 3 further comprises a GGPPS gene in combination with either ABS or TXS. Examples of GGPPS genes provide substrates for terpene synthase genes, i.e. ABS, or TXS. In some embodiments, terpene synthase genes are wild-type genes. In some preferred embodiments, terpene synthase genes contain mutations for producing variants of terpenoid products, as described and shown herein. In some embodiments, plasmid 3 further comprises a gene for terpenoid functionalizing enzymes, e.g., cytochromes P450.

[0426] In some preferred embodiments, plasmid 1 is under control of constitutive promoters. Thus, in some preferred embodiments, at least some of the genes that are part of the operon in plasmid 1 are constitutively expressed. In some preferred embodiments, at least some of the genes that are part of the operon in plasmid 1 are expressed when contacted with an inducible compound, i.e. under control of an inducible promoter, such as a lacZ promoter turned on when in contact with X-gal.

[0427] In some preferred embodiments, plasmids 2 and 3 are under control of inducible promoters. Thus, in some preferred embodiments, at least some of, and in some cases the entire set of genes contained in a metabolic pathway operon in plasmid 2 are expressed when contacted with an inducible compound. In some preferred embodiments, some genes expressed in plasmid 3 are under inducible control.

[0428] In some preferred embodiments, plasmid 4 is under the control of constitutive promoters. Thus, in some embodiments, at least one gene in plasmid 4 is under control of a constitutive promoter. In some embodiments, at least one gene in plasmid 4 is under control of an inducible promoter.

[0429] In some preferred embodiments, a host bacterium undergoes at least 2 rounds of transformation, e.g. first to transform plasmids 1 and 2 simultaneously into a strain that already harbors plasmid 4 (e.g., a S1030 strain which already comprises this accessory plasmid), followed by transformation with plasmid 3. In some preferred embodiments, a host bacterium undergoes at least 3 rounds of transformation, e.g. first to transfect plasmid 1, then transfect plasmid 2, followed by transfection of plasmid 3.

[0430] In some preferred embodiments, each plasmid has an antibiotic resistance gene (or other type of selective gene) for identifying successfully transformed bacteria for that plasmid, i.e. antibiotic resistance genes may be different for each plasmid. Thus, when an antibiotic resistance gene is expressed, instead of a bacteria stopped from normal replication when in contact with the antibiotic, a bacteria has resistance so is able to replicate at normal or near normal rates.

[0431] Thus, as described herein, laboratory stains of E. coli were engineered to comprise up to three types of expression plasmids by first transfecting with plasmid 1, then selecting for transformants (growing colonies) on/in antibiotic containing media wherein nontransformants do not grow, then transfecting transformants with plasmid 2 and selecting for double transformants, e.g. media containing antibiotics for allowing the growth of double transformants, then transfecting double transformants with plasmid 3 and selecting for triple transformants, e.g. media containing antibiotics for allowing the growth of triple transformants. In one embodiment, triple transformants are grown in media containing an inducer(s) for the inducible plasmids (2 and 3) in combination with the three antibiotics for producing products having at least some inhibitory activity for the chosen enzyme of plasmid 1, made by the enzymes provided by the combination of enzymes expressed by plasmids 2 and 3.

[0432] Further, as described herein, laboratory stains of E. coli were engineered to comprise up to four types of expression plasmids by first transforming host cells with plasmids 1 and 2, simultaneously, into a strain that already harbors plasmid 4, then selecting for triple transformants (growing colonies) on/in antibiotic containing media wherein non-transformants do not grow, then further transforming successful triple transformants with plasmid 3 and selecting for quadruple transformants, e.g. media containing antibiotics that allow for the growth of quadruple transformants. In one embodiment, quadruple transformants are grown in media containing (i) an inducer(s) for the inducible plasmids (2 and 3), (ii) a metabolic precursor for metabolite biosynthesis, e.g., mevalonate, and (iii) five antibiotics (i.e., one for each plasmid and one under control of the genetically encoded system in plasmid 1) for producing products having at least some inhibitory activity on the chosen enzyme of plasmid 1, made by the combination of enzymes expressed by plasmids 2 and 3.

[0433] In some embodiments, a terpenoid operon pathway intended for insertion into or already within plasmid 2, may be altered by swapping in a different gene for terpene synthases (i.e., in each row of FIG. 36, the metabolic pathway differs in the identity of the gene for a terpene synthase; when ADS or TXS are present, GGPPS is also present).

[0434] In FIGS. 41A, 41B, 41C, and 41D, for examples, we mutate (rather than swap) a single gene of a metabolic pathway: e.g. induce at least one mutation in a gene encoding amorphadiene synthase. After doing so, we show that a metabolic pathway can be mutated to generate a library of pathways, and that these pathways can be screened to identify pathways that generate more potent inhibitors of PTP1B than the unmutated parent pathway.

[0435] To summarize, we provided a demonstration that (i) the B2H system (detection operon) and (ii) a metabolic pathway for terpenoid biosynthesis can be combined within a host organism to identify genes involved with production of small-molecules and evolve genes related to production of small-molecules that may be inhibitors that enable the microbial synthesis of PTP1B inhibitors.

[0436] In preferred embodiments, small-molecule products are derived from one general metabolic pathway (the mevalonate-dependent pathway for terpenoid biosynthesis from Saccharomyces cerevisiae), and one host organism (Escherichia col). These small-molecule products produced as described herein, are contemplated for use as treatments of type 2 diabetes, obesity, and breast cancer, among other diseases.

[0437] Without being bound by theory, when a genetically encoded system for detecting the activity of a specified test enzyme is located within a host bacterium, a constitutive promoter expresses part A of the detection system (e.g. detection operon). So long as the phosphatase (or other test enzyme) expressed by part A is active, an expressed kinase enzyme, e.g. Src kinase, attaches a phosphate (P) group to the expressed second fusion protein comprising a substrate recognition domain (S) attached to a protein capable of recruiting RNA polymerase to DNA (e.g., the RP.sub.? subunit of RNA polymerase), and the phosphatase removes that phosphate group so that few molecules of phosphorylated fusion protein 2 stay bound to fusion protein 1 and, thus, few complexes between fusion proteins 2 and 1 form to initiate transcription of a gene of interest (GOI).

[0438] Thus, transcription of part B is off and the expression of a GOI is low, e.g. as observed when a GOI is a luminescent protein, so long as the placZ inducible promoter is not being induced. In this embodiment of an operon, the placZ inducible promoter is induced in order to allow the expression of a gene of interest in the absence of an inhibitor when not testing for inhibitor molecules.

[0439] However, in the presence of a small molecule that inhibits the phosphatase, a molecule either made endogenously from a metabolic pathway harbored by plasmids 2 and 3, or added to the growth media, then an excess of phosphorylated fusion protein 2 within the substrate binding region attaches to the substrate recognition domain of fusion protein 1 then when both are bound to the operator and the RB binding site then the GOI is expressed indicating the presence of a phosphatase inhibitor.

[0440] For practical purposes, it does not matter which fusion protein possesses a DNA-binding protein and which possesses a protein capable of recruiting RNA polymerase to DNA, so long as the DNA-binding protein constitutes part of one fusion protein and the protein that recruits RNA polymerase constitutes part of the other fusion protein, see FIG. 40, FIG. 10, for examples.

[0441] E. coli DH10B was used for molecular cloning and for preliminary analyses of terpenoid production; E. coli s1030.sup.1 was used for luminescence studies and for experiments involving terpenoid-mediated selection (e.g., molecular evolution); and E. coli B121 was used for experiments involving the heterologous expression and subsequent purification of proteins. However, it is not intended to limit the host bacteria strain to these E. coli strains. Indeed, any bacteria strain that supports the expression of the operons, DNA sequences and plasmids as described herein may be used as a host bacteria strain.

[0442] In preferred embodiments, small molecule products are derived from one general metabolic pathway (the mevalonate-dependent pathway for terpenoid biosynthesis from Saccharomyces cerevisiae), and one host organism (Escherichia coli). These small molecule products produced as described herein, are contemplated for use as treatments of type 2 diabetes, obesity, and breast cancer, among other diseases.

A. Bacterial Two-Hybrid (B2H) Systems (Operons) for the Identification of Microbially Synthesizable Inhibitors of PTP1B.

[0443] In one embodiment, an application of the B2H system to the evolution of genes that enable the microbial synthesis of molecules that (i) inhibit PTP1B and (ii) may be identified (i.e. structurally characterized) with standard analytical methods. In brief, the B2H system links the inactivation of PTP1B to the expression of a gene for antibiotic resistance. Accordingly, when a strain of E. coli (or other host bacterium) harbors both (i) the B2H system and (ii) a metabolic pathway for terpenoid biosynthesis, it will survive in the presence of antibiotics when it produces terpenoids that inhibit PTP1B.

[0444] A bacterial two-hybrid (B2H) system as described herein comprises one embodiment of an operon as described herein. Data displayed on left side of the plot in FIG. 33D (i.e., p130cas [also called Kras] and MidT substrates) is the same data displayed in FIG. 29A with the addition of providing more details of the B2H system in light of development.

[0445] We propose to use directed evolution to evolve new inhibitors; that is, we will manually introduce mutations into specific genes (or sets of genes) within a metabolic pathway to generate a library of metabolic pathways that can be screened alongside the B2H system. FIG. 4 1A describes a general approach to introduce mutations; Example C provides a very specific approach represented by FIG. 41A. To screen our library, we transform it into B2H-containing cells, and we grow them on plates containing various concentrations of spectinomycin; colonies that form on plates with high concentrations of spectinomycin contain a pathway capable of generating molecules that activate the B2H system (i.e., inhibit PTP1B). This pathway will not naturally evolve on its own. We can, thus, remove it from the first host cell, and transform it into another strain of E. coli to make high concentrations of inhibitors.

[0446] Embodiments of the system described herein enables the rapid identification of drug leads that can be readily synthesized in microbial hosts. It allows for a simultaneous solutions to two problems encountered during pharmaceutical development that are often examined separately 1) the identification of leads and 2) subsequent synthesis of those leads identified in 1).

[0447] Systems described herein have at least five uses: [0448] 1. Enables the identification of genes for proteins that generate inhibitors of the drug target. In brief, when the pathway for terpenoid biosynthesis generates target-inhibiting molecules, the cell survives at high antibiotic concentrations. By swapping out genes for terpene synthesizing and/or functionalizing enzymes, we can identify genes for enzymes that build such inhibitors. [0449] 2. Enables the construction of noveland, perhaps, unnatural-inhibitors. By mutating the pathway for terpenoid biosynthesis, we can generate pathways that confer survival at high antibiotic concentrations. These pathways contain mutated (i.e., unnatural) genes and, thus, can generate inhibitor molecules not found in Nature. [0450] 3. Enables the construction of inhibitors that overcome drug resistance. Briefly, after building a strain that generates a target-inhibiting molecule, we can carry out two steps: (i) We can mutate the drug target until it becomes resistant to that inhibitor. (ii) We can mutate the metabolic pathway until it generates an inhibitor of the mutated drug target. In this way, we can both (i) predict drug-resistance mutations and (ii) address those mutations by generating new inhibitors that overcome them. [0451] 4. Enables the construction of inhibitors of protein tyrosine kinases. Using a selection strategy similar to that described in 3.ii, we can mutate a metabolic pathway until it generates an inhibitor of Src kinase.
B. A Genetically Encoded System that Links the Inhibition of a Protein Tyrosine Phosphatase to Cell Survival.

[0452] In one preferred embodiment, a genetically encoded system was developed and used, as described herein, for detecting the presence of a small-molecule inhibitor of the catalytic domain of a chosen enzyme, e.g. a drug target enzyme, while allowing the survival of a host cell in the presence of a selective growth media. In other words, when the genetically encoded system is part of an expression plasmid in E. coli.

[0453] In one embodiment, an exemplary drug target enzyme was chosen, e.g. protein tyrosine phosphatase enzyme, protein tyrosine phosphatase 1B (PTP1B), In one embodiment, the genetically encoded system is part of an expression plasmid.

[0454] In one embodiment, the sensing operon is operably linked to a constitutive promoter for expression in E. coli.

[0455] FIG. 33A-E illustrates an embodiment of a genetically encoded system that links the activity of an enzyme to the expression of a gene of interest (GOI). Error bars in FIG. 33B-E denote standard deviation with n=3 biological replicates.

[0456] FIG. 33A illustrates an embodiment of a bacterial two-hybrid system that detects phosphorylation-dependent protein-protein interactions. Components include (i) a substrate domain fused to the omega subunit of RNA polymerase (yellow), (ii) an SH2 domain fused to the 434 phage cI repressor (light blue), (iii) an operator for 434cI (dark green), (iv) a binding site for RNA polymerase (purple), (v) Src kinase, and (vi) PTP1B. Src-catalyzed phosphorylation of the substrate domain enables a substrate-SH2 interaction that activates transcription of a gene of interest (GOI, black). PTP1B-catalyzed dephosphorylation of the substrate domain prevents that interaction; inhibition of PTP1B re-enables it. FIG. 33B refers to an embodiment of the two-hybrid system from FIG. 33A that (i) lacks PTP1B and (ii) contains luxAB as the GOI. We used an inducible plasmid to increase expression of specific components; overexpression of Src enhanced luminescence. FIG. 33C refers to an embodiment of the two-hybrid system from FIG. 33A that (i) lacks both PTP1B and Src and (ii) includes a superbinder SH2 domain (SH2*, i.e., an SH2 domain with mutations that enhance its affinity for phosphopeptides), a variable substrate domain, and LuxAB as the GOI. We used an inducible plasmid to increase expression of Src; luminescence increased most prominently for p130cas and MidT, suggesting that Src acts on both substrate domains. FIG. 33D refers to an embodiment of a two-hybrid system from FIG. 33C with one of two substrates: p130cas or MidT. We used a second plasmid to overexpress either (i) Src and PTP1B or (ii) Src and an inactive variant of PTP1B (C215S). The difference in luminescence between systems containing PTP1B or PTP1B(C215S) was greatest for MidT, suggesting that PTP1B acts on this substrate. Right: An optimized version of the two-hybrid system (with bb030 as the RBS for PTP1B) appears for reference. FIG. 33E displays the results of an exemplary growth-coupled assay performed using an optimized B2H including SH2*, a midT substrate, optimized promoters and ribosome binding sites (bb034 for PTP1B), and SpecR as the GOI. This system is illustrated at the top of the figure. Exemplary growth results demonstrate that inactivation of PTP1B enables strain of E. coli harboring this system to survive at high concentrations of spectinomycin (>250 ?g/ml).

1. Sequential Optimization of a Two-Hybrid System with LuxAB as the GOI.

[0457] Phase 1: We examined two different promoters for Src in a system that lacked PTP1B. Phase 2: We examined two different ribosome binding sites (RBSs) for Src in a system that lacked PTP1B. Phase 3: We examined two different RBSs for PTP1B in a complete system. Note: In phases 1 and 2, the operon contains wild-type (WT) or non-phosphorylate-able (mutant, Y/F) versions of the substrate domain. In phase 3, the operon contains wild-type (WT) or catalytically inactive (mutant, C215S) version so PTP1B. See, FIG. 34. FIG. 34 illustrates exemplary experiments used to optimize the B2H system depicted in FIG. 33.

2. Comparing RB Sites.

[0458] We grew strains of E. coli harboring versions of the bacterial two-hybrid that contained different RBSs for PTP1B (bb034 or bb030) on various concentrations of spectinomycin (left to right) and plated them on various concentrations of spectinomycin (top to bottom). We used bb034 for one embodiment of an optimized two-hybrid system shown in FIG. 33E. See, FIG. 35.

[0459] FIG. 35 FIG. 3 illustrates exemplary experiments used to optimize the B2H system depicted in FIG. 33 for growth-coupled assays. [0460] Rice, P., Longden, L. & Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 16, 276-277 (2000).

C. Biosynthesis of PTP1B-Inhibiting Terpenoids Enables Cell Survival.

[0461] When pTS contains ADS or GHS, it does not contain GGPPS; when pTS contains ABS or TXS, it also contains GGPPS; ABSD.sub.404A/D621A refers to a catalytically inactive variant of ABS; and B2H* contains PTP1B(C215S). ADS and, marginally, ABS enabled survival in the presence of spectinomycin, a result suggestive of the ability of these to terpene synthases to generate inhibitors of PTP1B.

[0462] FIG. 36A-C FIG. 4 shows an illustration of an operon (FIG. 36A) used for providing exemplary results during biosynthesis of PTP1B-inhibiting terpenoids FIG. 36B enabling cell survival FIG. 36C.

[0463] FIG. 36A-C FIG. 4 depicts an exemplary metabolic pathway for the biosynthesis of terpenoids.

[0464] FIG. 36A depicts a plasmid-borne pathway for terpenoid biosynthesis: (i) pMBIS, which harbors the mevalonate-dependent isoprenoid pathway of S. cerevisiae, converts mevalonate to isopentyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP). (ii) pTS, which encodes a terpene synthase (TS) and, when necessary, a geranylgeranyl diphosphate synthase (GPPS), converts IPP and FPP to sesquiterpenes and/or diterpenes.

[0465] FIG. 36B depicts exemplary terpene synthases: amorphadiene synthase (ADS) from Artemisia annua, 7-humulene synthase (GHS) from Abies grandis, abietadiene synthase (ABS) from Abies grandis, and taxadiene synthase (TXS) from Taxus brevifolia.

[0466] FIG. 36C shows the results of an exemplary growth-coupled assay of strain of E. coli that contains both (i) an embodiment of the optimized bacterial two-hybrid (B2H) system (i.e., the B2H system from FIG. 33E) and (ii) an embodiment of a pathway for terpenoid biosynthesis (i.e., the pathway from FIG. 35A).

[0467] Briefly, we grew strains of E. coli that harbored (i) the same pathway for producing linear isoprenoid precursors and (ii) a different plasmid encoding a terpene synthase (pTS). The pTS plasmid contained on of the following: (i) amorphadiene synthase (ADS) from Artemisia annua, (ii) 7-humulene synthase (GHS) from Abies grandis, (iii) abietadiene synthase (ABS) from Abies grandis in operable combination with a geranylgeranyl diphosphate synthase (GGPPS, (iv) taxadiene synthase (TXS) from Taxus brevifolia in operable combination with a GGPPS, (v) a inactive variant of ABS (i.e., ABSx, which corresponds to ABSD.sub.404A/D621A), or (vi) the L450Y mutant of GHS. After growing these strains, we compared the ability of their products to inhibit PTP1B by carrying out the following steps: (i) We used a hexane overlay to extract hydrophobic products (e.g., terpene-like products) from each culture, we then dried the products in a rotary evaporator, we dissolved the dried extract in dimethyl sulfoxide (DMSO), and we measured PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) in the presence and absence of extract-containing DMSO. We note: The L450Y mutant of GHS was included in our analysis because the wild-type form of GHS does not permit B2H-mediated growth in the presence of an antibiotic, but our preliminary data indicate that the L450Y mutant of GHS does permit such growth. Accordingly, we hypothesized that this mutant produced a molecule that is a stronger inhibitor of PTP1B than the molecules generated by wild-type GHS. See, FIG. 37A-C Demonstration of differential inhibition by structurally distinct terpenoids.

[0468] In examining FIG. 37A-C, we observed a trend: Extracts from strains containing terpene synthases that confer resistance to high concentrations of antibiotic (see FIG. 36) where ADS and GHS.sub.L450Y were more inhibitory than extracts from strains that did not confer resistance, e.g., TXS and ABSx. We note: strains containing ADS and GHS also included the optimized bacterial two-hybrid (B2H) system, but selection was not performed in the experiments used to product terpenoids for the experiments described by these figures.

[0469] FIG. 37A-C provides an exemplary analysis of the inhibitory effects of terpenoids generated by different strains of E. coli.

[0470] FIG. 37A depicts the results of our analysis of the inhibitory effect of DMSO containing (i) no inhibitor and (ii) extracted compounds from the culture broth of the ADS-containing strain.

[0471] FIG. 37B depicts the results of our analysis of the inhibitory effect of DMSO containing (i) extracted compounds from the culture broth of the GHS-containing strain (gHUM) or (ii) extracted compounds from the culture broth of the strain including the L450Y mutant of GHS.

[0472] FIG. 37C depicts the results of our analysis of the inhibitory effect of DMSO containing (i) no inhibitor, (ii) extracted compounds from the culture broth of the ABS-containing strain, (iii) extracted compounds from the culture broth of the TXS-containing strain, and (iv) extracted compounds from the culture broth of the train strain containing a catalytically inactive variant of ABS.

[0473] Briefly, we grew strains of E. coli containing both (i) the optimized bacterial two-hybrid system and (ii) a terpenoid pathway with mutants ?-humulene synthase (GHS; 1 mutant/cell) on varying concentrations of spectinomycin. Above: product profiles of strains with GHS mutants that conferred survival at high antibiotic concentrations. See, FIG. 38.

[0474] FIG. 38 shows exemplary analysis of the product profiles of mutants of GHS that enabled growth in the presence of spectinomcyin.

[0475] In brief, we constructed versions of the bacterial two-hybrid system that include SH2*, the midT substrate, optimized promoters and ribosome binding sites, SpecR, and alternative PTPs: the catalytic domain of PTPN6 (e.g., SHP-1) and PTP1B.sub.405 (the full-length version of PTP1B). Note: these systems are identical to the B2H system depicted in FIG. 33E, except they possess only one of the following PTP genes: PTP1B (as in FIG. 33E), PTPN6 (different from FIG. 33E), or full-length PTP1B. Inactivation of the catalytic domain of both PTPN6 and the full-length PTP1B enabled strains of E. coli harboring corresponding operons to survive at high concentrations of spectinomycin (>400 ?g/ml). To extend our operon to other PTPs, we plan on modifying the substrate, SH2, and/or kinase domains. See, FIG. 39.

[0476] FIG. 39 An analysis of exemplary B2H systems that link the inhibition of other PTPs to cell survival.

[0477] We also generated versions of the bacterial two-hybrid system that include SH2*, the midT substrate, optimized promoters and ribosome binding sites, SpecR, and alternative PTPs: the catalytic domain of PTPN6 (e.g., SUP-1) and PTP1B.sub.405 (the full-length version of PTP1B).

[0478] Inactivation of the catalytic domain of PTPN6 and the full-length PTP1B enabled strains of E. coli harboring corresponding operons to survive at high concentrations of spectinomycin (>400 g/ml). To extend our operon to other PTPs, we plan on modifying the substrate, SH2, and/or kinase domains.

[0479] FIG. 40A-E depicts exemplary embodiments of genetically encoded systems that link the activity of an enzyme to the expression of a gene of interest, and the application of those embodiments to (i) the prediction of resistance mutations, (ii) the construction of inhibitors that combat resistance mutations, and (ii) the evolution of inhibitors of kinases.

[0480] FIG. 40A depicts an exemplary first step in examining potential resistance mutations. By evolving a metabolic pathway to produce molecules that inhibit a known drug target (e.g., PTP1B); these molecules will permit expression of a gene of interest (GOI) that confers survival in the presence of a selection pressure (e.g., the presence of spectinomycin, an antibiotic). FIG. 40B depicts an exemplary second step in examining potential resistance mutations. In a second strain of E. coli, we will replace the original gene of interest with a second (GOI2) that confers conditional toxicity (e.g., SacB, which converts sucrose to levan, a toxic product); we will evolve the drug target to become resistant to the endogenous inhibitors, while still retaining its activity. This mutant will prevent expression of the toxic gene. FIG. 40C depicts an exemplary third step in combating resistance mutations. In a third strain of E. coli, we will evolve a metabolic pathway that produces molecules that inhibit the mutated drug target. In this way, we will both predictand, through our second evolved pathway, address-mutations that might cause resistance to terpenoid-based drugs. We note: FIG. 40A-40C describe the use of our genetically encoded system to evolve inhibitors, but the steps 2 and 3 could be used to predict mutations that permit resistance to endogenously supplied inhibitors and, subsequently, to identify new endogenously supplied inhibitors that might combat that resistance. FIG. 40D depicts an exemplary genetically encoded system that detects inhibitors of an Src kinase. In brief, Src activity enables expression of a toxic gene (GOI2); inhibition of Src, in turn, would confer survival.

[0481] One embodiment of a configuration of the B2H architecture that enables survival when PTP1B is active, that is, when the activity of Src kinase is successfully canceled out. In the absence of PTP1B, this configuration could be used to evolve inhibitors of Src kinase; such an inhibitor would act similarly to PTP1B by preventing the phosphorylation of the substrate domain (as shown in FIG. 40E). Src kinase is a validated drug target; tyrosine kinases are targets of over 40 FDA-approved drugs.

[0482] FIG. 40E demonstrates one embodiment of a roof of principle for the B2H system describe in FIG. 40B. The system shown here includes two GOIs: SpecR and SacB. Expression of the GOIs confers survival in the presence of spectinomycin; expression of the GOIs causes toxicity in the presence of sucrose. The images depict the results of a growth-coupled assay performed on a strain of E. coli in the presence of various concentrations of sucrose. The strain harboring an active form of PTP1B (WT) grows better at high sucrose concentrations that the strain harboring an inactive form of PTP1B (C215S).

[0483] FIG. 41A depicts an exemplary strategy for the evolution of inhibitors of PTP1B.

[0484] FIG. 41A depicts an exemplary structural analysis used to identify targets for mutagenesis in the active sites of terpene synthases. It shows an alignment of the class I active site of ABS (gray, PDB entry 3s9v) and TXS (blue, PDB entry 3p5r) with the locations of sites targeted for site-saturation mutagenesis (SSM) highlighted on ABS (red). A substrate analogue (yellow) of TXS appears for reference. FIG. 41B depicts an exemplary strategy for introducing diversity into libraries of metabolic pathways: An iterative combination of SSM of key sites on a terpene synthase (as in a), error-prone PCR (ePCR) of the entire terpene synthase gene, SSM of key sites on a terpene-functionalizing enzyme (e.g., P450), and ePCR of the entire terpene-functionalizing enzyme. FIG. 41C depicts an exemplary quantification the total terpenoids present in DMSO samples with extracts of various TS-containing strains. In brief, we performed site-saturation mutagenesis of six sites on ADS (analogous to the sites shown in a); we plated the SSM library on agar plates containing different concentrations of spectinomycin; we picked colonies that grew on a plate containing a high concentration (800 ?g/ml) of spectinomycin and used each colony to inoculate a separate culture; we used a hexane overlay to extract the terpenoids secreted into each culture broth; we dried the hexane extract in a rotary evaporator and re-suspended the solid in DMSO; and we used a GC-MS to quantify the total amount of terpenoids present in the DMSO.

[0485] ADS WT, ADS F514E, ADS F370L, ADS 400A, ADS G439A, and ADS G400L describe mixtures of molecules generated by strains of E. coli harboring mutants of amorphadiene synthase (ADS). The labels describe the mutant: G439A corresponds to a mutant of abietadiene synthase in which glycine 439 has been mutated to alanine, and so on. In future work, we plan on (i) purifying different terpenoids from these mixtures, (ii) assessing their inhibitory effect on PTP1B in vitro, (iii) assaying their inhibitory effect on other PTPs (notably TC-PTP and PTPN11) in vitro, and (iv) assaying their influence on mammalian cells. See, FIG. 41D.

[0486] FIG. 41D depicts an exemplary analysis of the inhibitory effect of various extracts on PTP1B. In brief, the figure shows initial rates of PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) in the presence of terpenoids quantified in FIG. 41C. Two mutants of ADS (G439A and G400L) generate particularly potent inhibitors of PTP1B.

[0487] FIG. 42 depicts an exemplary analysis of the link between B2H activation and cell survival. An exemplary strain of E. coli that contains both (i) the optimized bacterial two-hybrid (B2H) system (FIG. 33E) and (ii) the terpenoid pathway depicted in FIG. 36A. Note: pTS includes GGPPS only when ABS or TXS are present; the Y/F operon corresponds to a B2H system in which the substrate domain cannot be phosphorylated. Survival at high concentrations of spectinomycin requires activation of the B2H system (i.e., phosphorylation of the substrate domain, a process facilitated by inhibition of PTP1B).

[0488] FIG. 43 provides exemplary product profiles of strains of E. coli harboring various terpene synthases. For this figure, the strain of E. coli harbored (i) the optimized B2H system (FIG. 33E) and (ii) the terpenoid pathway (FIG. 36A). The pathways corresponding to each profile differ only in the composition of the pTS plasmid, which contains TXS (taxadiene synthase from Taxus brevifolia and a geranylgeranyl diphosphate synthase from Taxus Canadensis); GHS (7-humulene synthase from Abies grandis); ADS (amorphadiene synthase from Artemisia annua); ABS (abietadiene synthase from Abies grandis and a geranylgeranyl diphosphate synthase from Taxus Canadensis); G400A (the G400A mutant of amorphadiene synthase from Artemisia annua); and G439L (the G439L mutant of amorphadiene synthase from Artemisia annua). Note that the two mutants of ADS yield different product profiles than the wild-type enzyme (ADS); our results indicate that products generated by these two mutants are more inhibitory than those generated by the wild-type enzyme (FIG. 41E).

D. Identification of Sites for Site Saturation Mutagenesis (SSM).

[0489] The active sites of terpene synthases and cytochrome P450s contain constellations of amino acids that guide catalysis in two ways: (i) They control the conformation space available to reacting substrates, and (ii) they alter the organization of water that surrounds substrates.sup.8-10. We identified plastic residues likely to modulate these attributes in the class I active sites of terpene synthase by carrying out the following steps: (i) We aligned the crystal structure of ABS with the crystal structure of TXS. (ii) We selected all residues within 8 angstroms of the substrate analog (2-fluoro-geranylgeranyl diphosphate) of the class I active site of TXS, and we identified a subset of sites that differed between ABS and TXS. (iii) We aligned the sequences of ABS,

[00002]$\text{?}=\frac{\text{?}}{\text{?}}+\frac{\text{?}}{\text{?}}$$\text{?}\text{indicates text missing or illegible when filed}$

[0490] GHS, delta-selenine synthase (DSS), and epi-isozizaene synthase (EIS). (iv) We used Eq. S1 to score each site based on its variability in size and hydrophilicity across the five enzymes analyzed. In this equation, ?.sub.V.sup.s is the variance in volume, ?.sub.HW.sup.s is the variance in Hopp-Woods index, and n.sub.v and n.sub.HW are normalization factors (based on the highest variances measured in this study). (v) We ranked each site according to S and selected the six highest-scoring sites. We note: For this analysis, we chose ABS and TXS because they are structurally similar enzymes (i.e., both possess ?, ?, and ? domains) with crystal structures; we chose GHS, DSS, and EIS because they have been shown to exhibit mutation-responsive product profiles.

[0491] FIG. 44A-D provides exemplary structural and sequence-based evidence that supports the extension the B2H system to other protein tyrosine phosphatases (PTPs).

[0492] FIG. 44A provides an exemplary structural alignment PTP1B and PTPN6, two PTPs that are compatible with the B2H system (see FIGS. 1e and 7 of Update A for evidence of compatibility). We used the align function of PyMol to align each structure of PTPN6 with either (i) the ligand-free (3A5J) or (ii) ligand-bound (2F71) structure of the catalytic domain of PTP1B. The align function carries out a sequence alignment followed by a structural superposition and, thus, effectively aligns the catalytic domains of both proteins. FIG. 44B provides an exemplary structural comparison of PTP1B and PTPN6; the root-mean-square deviations (RMSD) of aligned structures of PTP1B and PTPN6 range from 0.75 to 0.94 ?. FIG. 44C proves an exemplary sequence alignment of the catalytic domains of PTP1B (SEQ ID NO: 3) and PTPN6 (SEQ ID NO: 4) (EMBOSS Needle.sup.1). FIG. 44D provides an exemplary sequence comparison of the catalytic domains of PTP1B and TPPN6. The sequences share 34.1% sequence identity and 53.5% sequence similarity. In summary, the results of this figure indicate that our B2H system can be readily extended to PTPs that possess catalytic domains that are (i) structurally similar to the catalytic domain of PTP1B (here, we define structural similarity as two structures that when aligned, have with an RMSD of ?0.94 ? RMSD with the framework similar to the one used by the align function of PyMol) and/or (ii) sequence similar to the catalytic domain of PTP1B (here, we define sequence similarity as ?34% sequence identity or ?53.5% sequence similarity as defined by the EMBOSS Needle algorithm).

[0493] To identify plastic residues capable of adjusting the activity of P450BM3, we carried out an approach similar to that described above: (i) We used the mutant database (http://www.MuteinDB.org) to identify the 25 most commonly mutated sites in functional variants of P450.sub.BM3. (ii) We used Eq. S1 to score each site based on its variability in size and hydrophobicity across different mutants. (iii) We ranked each site according to S and selected the 7 highest-scoring sites. Site S1024 scored highly based on S but was omitted due to its location on the P450 reductase domain.

E. Exemplary Purification of Products.

[0494] See section relating to flash chromatography and HPLC.sup.1-3.

F. Exemplary Concentration Range for Testing Products.

[0495] We plan on incubating mammalian cells with 1-400 ?M of inhibitors; we will assess the biochemical influence of those inhibitors by using the assays described below.

G. Exemplary Cell-Based Assays.

[0496] We will characterize the biological activity of newly developed inhibitors in at least two ways: [0497] 1. We will assay the influence of inhibitors on insulin receptor phosphorylation. In brief, we will expose HepG2, Hela, Hek393t, MCF-7, and/or Cho-hIR cells to insulin shock in the presence and absence of inhibitors, and we will use a western blot and/or an enzyme-linked immunosorbent assay (ELISA) to measure the influence of the inhibitors on insulin receptor phosphorylation. In some embodiments we may use cell-permeable inhibitors of PTP1B to enhance insulin receptor phosphorylation. [0498] 2. We will examine the morphological and/or growth effects inhibitors identified in a system described herein on cellular models of HER2(+) and TN breast cancer. In brief, we will examine the relevance of inhibitors to HER2(+) breast cancer by evaluating their ability to inhibit the migration of BT474 and SKBR3 cells, which are HER2(+), but not MCF-7 and MDA-MB-231 cells, which are HER2(?). We will examine the relevance of inhibitors to triple negative breast cancer, in turn, by carrying out viability and proliferation assays on panels of TN cell lines (e.g., ATCC TCP-1002). All cell lines are available from the ATCC (ATCC.org) and have been used previously to characterize potential therapeutics for HER2(+) and TN subtypes.sup.4,5.

[0499] It is not meant to limit a pathway to terpenoid synthesis. Indeed, an alkaloid biosynthesis pathway is contemplated for use to identify,

[0500] An exemplary pathway for alkaloid biosynthesis consists of three modules (Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. (2011). doi:10.1038/ncomms1327, herein incorporated by reference) (i) the first enables the overexpression of our enzymes for L-tyrosine overproduction: TKT, PEPS, fbr-DAHPS, and fbr-CM/PDH; (ii) the second enables the expression of three enzymes necessary for the construction of dopamine and 3,4-DHPAA: TYR, DODC, and MAO; and (iii) the third enable the expression of four enzymes for the construction of (S) reticuline from 3,4-DHPAA and dopamine: NCS, 60MT, CNMT, and 4OMT. Enzymes are as follows: TKT, transketolase (tktA, GenBank accession number X68025); PEPS, phosphoenolpyruvate (PEP) synthetase (ppsA, GenBank accession number X59381); fbr-DAHPS, feedback-inhibition resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr, GenBank accession number J01591); fbr-CM/PDH, feedback-inhibition resistant chorismate mutase/prephenate dehydrogenase (tyrAfbr, GenBank accession number M10431); TYR, tyrosinase of Streptomyces castaneoglobisporus (ScTYR containing tyrosinase and its adaptor protein, ORF378, GenBank accession numbers AY254101 and AY254102); DODC, DOPA decarboxylase of Pseudomonas putida (GenBank accession number AE015451); MAO, monoamine oxidase of Micrococcus luteus (GenBank accession number AB010716); NCS, norcoclaurine synthetase of C. japonica (GenBank accession number AB267399); 60MT, norcoclaurine 6-O-methyltransferase of C. japonica (GenBank accession number D29811); CNMT, coclaurine-N-methyltransferase of Coptis japonica (GenBank accession number AB061863); 4OMT, 3-hydroxy-N-methylcoclaurine 4-O-methyltransferase of C. japonica (GenBank accession number D29812). We note; these three modules may be encoded by two plasmids.

References for Section V, Herein Incorporated by Reference in their Entirety

[0501] 1. Jia, M., Potter, K. C. & Peters, R. J. Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis. Metab. Eng. 37, 24-34 (2016). [0502] 2. Criswell, J., Potter, K., Shephard, F., Beale, M. H. & Peters, R. J. A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase. Org. Lett. 14, 5828-5831 (2012). [0503] 3. Morrone, D. et al. Increasing diterpene yield with a modular metabolic engineering system in E. coli: Comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85, 1893-1906 (2010). [0504] 4. Dagliyan, O. et al. Engineering extrinsic disorder to control protein activity in living cells. Science (80-.). 354, 1441-1444 (2016). [0505] 5. Lehmann, B. D. et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J. Clin. Invest. (2011). doi:10.1172/JCI45014 [0506] 6. Dempke, W. C. M., Uciechowski, P., Fenchel, K. & Chevassut, T. Targeting SHP-1, 2 and SHIP Pathways: A novel strategy for cancer treatment? Oncology (Switzerland) (2018). doi:10.1159/000490106 [0507] 7. Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. (2011). doi:10.1038/ncomms1327 [0508] 8. Christianson, D. W. Structural biology and chemistry of the terpenoid cyclases. Chem. Rev. 106, 3412-3442 (2006). [0509] 9. Fasan, R. Tuning P450 enzymes as oxidation catalysts. ACS Catalysis 2, 647-666 (2012). [0510] 10. Jung, S. T., Lauchli, R. & Arnold, F. H. Cytochrome P450: Taming a wild type enzyme. Current Opinion in Biotechnology 22, 809-817 (2011). [0511] 11. Braun, A. et al. MuteinDB: The mutein database linking substrates, products and enzymatic reactions directly with genetic variants of enzymes. Database (2012). doi:10.1093/database/bas028

VI. Evolving Optogenetic Actuators: Photoswitchable Constructs

[0512] A. Optical Control with Red and Infrared Light.

[0513] Contemporary efforts for using light to control enzyme activity have relied on at least two optogenetic actuators: LOV2, which has terminal helices that are destabilized by blue light (?450 nm).sup.2,18,48, and Dronpa, which switches from a dimer to a monomer in response to green light (?500 nm).sup.19. Unfortunately, blue and green light suffer from problems of phototoxicity, penetration depth, and spectral similarity that limit their use in signaling studies.sup.21. Thus, in one embodiment, photoswitchable enzymes stimulated by red or infrared light are contemplated for development. These wavelengths have lower phototoxicities and greater penetration depths than blue and green light.sup.20,21, and will permit multi-color actuation alongside blue or green light.

B. An Operon to Evolve Photoswitchable Constructs.

[0514] In one embodiment, an operon that links the activity of PTP1B to cell growth is contemplated. In brief, this operon is based on the following control strategy (some additional details in FIG. 10): A kinase stimulates the binding of two proteins, which in turn, promote transcription of an essential gene; PTP1B suppresses the binding of these two proteins and, thus, inhibits transcription. This operon allows cells in possession of photoswitchable variants of PTP1B to grow faster in the presence of one light source than in the present of another (e.g., 750 nm vs. 650 nm). The difference in growth rates enables the identification of functional chimeras. Initial experiments with an operon based on Lux-based luminescence (based on a system developed by Liu and colleagues.sup.53) show a 20-fold difference in luminescence between a strain expressing two model binding partners and a strain expressing one (FIG. 3E). We will continue to develop this operon by adding a protein-protein interaction that is modulated by a PTP and PTK (see below).

[0515] This operon allows cells in possession of photoswitchable variants of PTP1B to grow faster in the presence of one light source than in the present of another (e.g., 750 nm vs. 650 nm). The difference in growth rates enables the identification of functional chimeras. Initial experiments with an operon based on Lux-based luminescence (based on a system developed by Liu and colleagues 53) show a 20-fold difference in luminescence between a strain expressing two model binding partners and a strain expressing one (FIG. 3E). We will continue to develop this operon by adding a protein-protein interaction that is modulated by a PTP and PTK.

[0516] FRET sensors. We will use Forster resonance energy transfer (FRET) to monitor the activity of PTP1B in living cells. Our preliminary sensor exhibits a 20% reduction in FRET signal when treated with Src kinase (FIG. 3F). Previous imaging studies indicate that a 20% change in FRET is sufficient to monitor intracellular kinase activity5456. To enhance spatial resolution in imaging studies, we will attempt to optimize our sensor further (and use it to measure the activity of PTP1B in vitro).

1. To Evolve Phosphatases and Kinases Modulated by Red and Infrared Light.

[0517] This section uses directed evolution to build enzymes that can be turned on and off with red and infrared light. We will know that we are successful when we have (i) built a genetic operon that links the activity of PTP1B to antibiotic resistance, (ii) A used that operon to build a PTP1 B-phytochrome chimera that exhibits a three- to ten-fold change in activity in response to red and infrared light, and (iii) built similar phytochrome chimeras of STEP and PTK6.

[0518] Hypothesis. Phytochrome proteins exhibit global conformational changes when exposed to red and infrared light.sup.27,2.sup.8, but to date, have eluded rational integration into photoswitchable enzymes. We hypothesize that a genetic operon that links PTP or PTK activity to cell growth will enable the evolution of PTP- or PTK-phytochrome chimeras stimulated by red or infrared light.

[0519] Experimental approach: We will build an operon that links PTP1B inhibition to antibiotic resistance, and we will use that operon to evolve photoswitchable PTP1 B-phytochrome chimeras. This effort will involve (i) the construction a library of PTP1 B-phytochrome chimeras that differ in linker composition and/or linker length, (ii) the use of our operon to screen that library for functional mutants, (iii) a kinetic and biostructural characterization of the most photoswitchable mutants, and (iv) the extension of this approach to STEP and PTK6. This effort has two major goals: a variant of PTP1B modulated by red and/or infrared light, and a general approach for using directed evolution to extend optical control to new enzymes and different wavelengths of light.

2. Development of a Synthetic Operon for Evolving PTP1 B-Phytochrome Chimeras.

[0520] We will build a variant of PTP1B that can be modulated by red and infrared light by attaching its C-terminal ?-helix to the N-terminal ?-helix of bacterial phytochrome protein 1 (BphP1) from Rhodopseudomonas palustris (FIG. 9); this protein undergoes a reversible conformational change when exposed to 650 nm and 750 nm light. Phytochromes such as BphP1 are valuable for photocontrol because they can be actively toggled between conformations (i.e., turned on and off). Their structures, however, are not compatible with cage-based actuation (they do not undergo large-scale unwinding); they have, thus, been overlooked in previous efforts to develop photoswitchable enzymes.

[0521] We will evolve photoswitchable PTP1B-BphP1 chimeras by using a genetic operon that links PTP1B activity to antibiotic resistance. This operon will consist of six components (FIG. 10A-B): (i) a PTP1B substrate domain tethered to a DNA-binding protein, (ii) a substrate recognition domain (i.e., a substrate homology 2 domain, or SH2) tethered to the subunit of an RNA polymerase, (iii) an Src kinase (a kinase capable of phosphorylating a wide range of substrates), (iv) PTP1B (or a potentially photoswitchable variant of PTP1B), (v) a gene for antibiotic resistance, and (vi) an operator for that gene.

[0522] With this system, light-induced inactivation of PTP1B will enable transcription of the gene for antibiotic resistance. Previous groups have used similar operons to evolve protein-protein binding partners (our system is based on an operon used by Liu et al. to evolve insecticidal proteins.sup.53); here, we take the additional (new) steps of (i) using a protein-protein interaction mediated by enzymes (phosphatases and kinases) and (ii) screening that interaction in the presence and absence of light.

[0523] We have begun to develop our operon by using a Lux-based luminescence as an output. Preliminary results show that model protein-protein binding partners can elicit a 20-fold change in luminescence (FIG. 3E). We plan to swap out these binding partners with substrate and SH2 domains, and test the new system alongside simultaneously expressed PTP1B and Src kinase (which have some complementary activities, and can be expressed in E. coli.sup.68,69)

[0524] Advantages of using operons expressing photosensitive phosphatases includes but is not limited to enabling high-throughput screens of mutants of photoswitchable enzymes and provides a method for screening the libraries of enzymes that they motivate, see, FIG. 5A for example. In contrast, have shown that mutagenesis a photoswitchable enzyme can adjust (i.e., improve) its dynamic range (i.e., ratio of dark-state activity to light-state activity); while some published studies, such as WO2011002977. Genetically Encoded Photomanipulation Of Protein And Peptide Activity. Published Jan. 6, 2011, have proposed, but not shown, that mutagenesis of protein light switches might enable spectral tuning of photoswitchable enzymes. WO2011002977, provides a list of sites that could be mutated to modify the flavin-binding pocket of LOV2 to accept flavins that absorb light at alternative wavelengths. However, their construct is described as a LOV2 domain of Avena sativa (oat) phototropin 1 (404-546), including the C-terminal helical extension J alpha where J? unwinds instead of the A alpha helix described herein. Nonetheless, there is no available method for carrying out high-throughput screens of mutants with modified binding pockets for which the invention described herein provides a platform for doing so. Further, in contrast to WO2013016693. Near-infrared light-activated proteins. Publication Date 01.31.2013, inventions described herein provide a platform for screening potentially improved/modified variants of photoswitchable proteins, such as a plant phototropin 1 LOV2.

[0525] Additionally, methods for screening the libraries of enzymes enable the detection of (i) molecules or (ii) photoswitchable domains that change the activity of any enzyme that, in turn, can modulate the affinity, or outcome associated with, a protein-protein interaction: protein tyrosine phosphatase (PTPs) and protein tyrosine kinases (PTKs) are demonstrated. Moreover, proteases are contemplated as proteins to add to this system.

C. Directed Evolution.

[0526] We will build libraries of PTP1B-BphP1 chimeras by pairing overlap extension PCR (oePCR) with error-prone PCR (epPCR). Specifically, we will use oePCR to build chimeras that differ in linker length (here, we define the linker as the ?20 residue region comprised of the C-terminal ?-helix of PTP1B and the N-terminal ?-helix of BphP1), and we will use epPCR to vary linker composition. Depending on the results of this initial library, we may extend error-prone PCR into the BphP1 gene, but we will not mutate PTP1B beyond its C-terminal ?-helix.

[0527] In the presence of a small amount of antibiotic (i.e. an amount that impedes the growth of E. coli), our genetic operon will cause cells that contain functional PTP1B-BphP1 chimeras to exhibit different growth rates under red and infrared light. We will exploit these differences to identify cells that harbor photoswitchable constructs. In brief, we will (i) generate two replicate plates of cell colonies, (ii) grow one under red light and one under infrared light (FIG. 11 A), and (iii) select a subset of colonies (top hits) that show differential growth. We will further characterize our top hits by growing them in small-scale liquid cultures (e.g., 96-well plates with ?1 ml/well; FIG. 11B) under red and infrared light, and by sequencing the PTP1 B-BphP1 genes of colonies that show the greatest different in growth rates.

[0528] We will attempt to build enzyme-phytochrome chimeras of STEP and PTK6 by pursuing two strategies: (i) We will replace PTP1B in our final PTP1B-BphP1 chimera with STEP or PTK6; this strategy will allow us to assess the modularity of our final design, (ii) We will use our operon-based approach to evolve functional STEP-BphP1 and PTK6-BphP1 chimeras; this strategy will allow us to assess the generalizability of our approach to evolution.

[0529] Operons for evolving STEP-BphP1 and PTK6-BphP1 chimeras will closely resemble the PTP IB-specific operon. For STEP, we will use a STEP-specific substrate and SH2 domain (Src kinase, which has a broad substrate specificity, is likely to have complementary activities on a subset of STEP substrates); for PTK6, we will use a recognition process that is inhibitednot activatedby phosphorylation (here, we can use PTP1B.sub.WT as the complementary enzyme).

D. Extension of Approach.

[0530] We will attempt to build enzyme-phytochrome chimeras of STEP and PTK6 by pursuing two strategies: (i) We will replace PTP1B in our final PTP1B-BphP1 chimera with STEP or PTK6; this strategy will allow us to assess the modularity of our final design, (ii) We will use our operon-based approach to evolve functional STEP-BphP1 and PTK6-BphP1 chimeras; this strategy will allow us to assess the generalizability of our approach to evolution.

[0531] Operons for evolving STEP-BphP1 and PTK6-BphP1 chimeras will closely resemble the PTP IB-specific operon. For STEP, we will use a STEP-specific substrate and SH2 domain (Src kinase, which has a broad substrate specificity, is likely to have complementary activities on a subset of STEP substrates); for PTK6, we will use a recognition process that is inhibitednot activatedby phosphorylation (here, we can use PTP1B.sub.WT as the complementary enzyme)

F. Exemplary Contemplated Characterization: Biophysical Characterization of Enzyme-Phytochrome Chimeras.

[0532] We will examine the structural basis of photocontrol in the most photoswitchable chimeras by using a subset of crystallographic and kinetic analyses. X-ray crystal structures will show how BphP1 affects the structures of PTP1B, STEP, and PTK6. Kinetic studies will show how BphP1 affects substrate specificity and binding affinity (or more specifically, K.sub.m, which is affected by binding affinity).

TABLE-US-00003 TABLE1 ExemplaryPromoters. SEQID Name DNASequence NO.# Pro1 TTCTAGAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTA SEQIDNO. TGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGGTATCTATA 25 TTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTTT ACTAGAG ProD GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGG SEQIDNO. TTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTATAATATATTCAGGG 26 AGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTTTACTAGAG pBAD AGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTA SEQIDNO. CTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCT 27 GTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTC TATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTT TGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGC TTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGC pLacZOpt ACAAGAAAGTTTGTTCATTAGGCACCCCGGGCTTTACTCGTAAAGCTTCC SEQIDNo. (cIoperate GGCGCGTATGTTGTGTCGACCG 28 bolded) pTrc CGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCT SEQIDNo. GTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAG 29 GCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCT GGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAA TGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG T7 CCTATAGTGAGTCGTATTA SEQIDNo. 30

TABLE-US-00004 TABLE2 ExemplaryRibosomeBindingSites. SEQID Name DNASequence NO.# proRBS TTAAAGAGGAGAAAGGTC SEQID NO.31 Sal28RBS CGAAAAAAAGTAAGGCGGTAATCC SEQID NO.32 bb034RBS TGCAGAAAGAGGAGAAATACTAG SEQID NO.33 bb030 ATTAAAGAGGAGAAATACTAG SEQID No.34 RBSforGOI GTGCAGTAAGGAGGAAAAAAAA SEQID inB2H No.35 bbAH GCTAGCTTTAAGAAGGAGATATACC SEQID No.36

TABLE-US-00005 TABLE3 ExemplaryProteinSequences(includestruncations). SEQID Name AminoAcidSequence NO.# RpoZ(linker MARVTVQDAVEKIGNRFDLVLVAARRARQMQVGGKDPLVPEENDKTTV SEQID bolded) IALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGRRAAA NO.37 cI(linkerbolded) MSISSRVKSKRIQLGLNQAELAQKVGTTQQSIEQLENGKTKRPRFLPELAS SEQID ALGVSVDWLLNGTSDSNVRFVGHVEPKGKYPLISMVRARSWCEACEPYD NO.38 IKDIDEWYDSDVNLLGNGFWLKVEGDSMTSPVGQSIPEGHMVLVDTGRE PVNGSLVVAKLTDANEATFKKLVIDGGQKYLKGLNPSWPMTPINGNCKII GVVVEARVKFVDYKDDDDK SH2 WYFGKITRRESERLLLNPENPRGTFLVRESETVKGAYALSVSDFDNAKGL SEQID NVKHYLIRKLDSGGFYITSRTQFSSLQQLVAYYSKHADGLCHRLTNVC NO.39 KrasSubstrate WMEDYDYVHLQG SEQID NO.40 MidTSubstrate EPQYEEIPIYL SEQID NO.41 ShcASubstrate DHQYYNDFPG SEQID NO.42 EGFRSubstrate PQRYLVIQGD SEQID NO.43 Src MSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAI SEQID KTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKG NO.44 SLLDFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANIL VGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKS DVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHD LMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL CDC37 MVDYSVWDHIEVSDDEDETHPNIDTASLFRWRHQARVERMEQFQKEKEE SEQID LDRGCRECKRKVAECQRKLKELEVAEGGKAELERLQAEAQQLRKEERSW NO.45 EQKLEEMRKKEKSMPWNVDTLSKDGFSKSMVNTKPEKTEEDSEEVREQK HKTFVEKYEKQIKHFGMLRRWDDSQKYLSDNVHLVCEETANYLVIWCID LEVEEKCALMEQVAHQTIVMQFILELAKSLKVDPRACFRQFFTKIKTADR QYMEGFNDELEAFKERVRGRAKLRIEKAMKEYEEEERKKRLGPGGLDPV EVYESLPEELQKCFDVKDVQMLQDAISKMDPTDAKYHMQRCIDSGLWVP NSKASEAKEGEEAGPGDPLLEAVPKTGDEKDVSV PTP1B MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDV SEQID SPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMV NO.46 WEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISED IKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHSSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLL EMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEP PPEHIPPPPRPPKRILEPHN MBP MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFP SEQID QVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA NO.47 VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKS ALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGL TFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKV NYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMS AFWYAVRTAVINAASGRQTVDEALKDAQTRITK LuxAB MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHHFTEF SEQID GLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQM NO.48 SKGRFRFGICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYM EADNEHIKFHKVKVNPAAYSRGGAPVYVVAESASTTEWAAQFGLPMIL SWIINTNEKKAQLELYNEVAQEYGHDIHNIDHCLSYITSVDHDSIKAKEIC RKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFVLKGHKDTNRR IDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASMKLFQ SDVMPFLKEKQRSLLYYGGGGSGGGGSGGGGSGGGGSKFGLFFLNFINS TTVQEQSIVRMQEITEYVDKLNFEQILVYENHFSDNGVVGAPLTVSGFLL GLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGFSDCEKKDEMH FFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYTPG GPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKY DVDLSEIDHQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLE EIIAENAVGNYTECITAAKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIV DDNIKKYHTEYT SpecR MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDL SEQID LVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRY NO.49 PAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAE ELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTG KIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEE FVHYVKGEITKVVGK AgAs MVKREFPPGFWKDDLIDSLTSSHKVAASDEKRIETLISEIKNMFRCMGY SEQID GETNPSAYDTAWVARIPAVDGSDNPHFPETVEWILQNQLKDGSWGEG NO.50 FYFLAYDRILATLACIITLTLWRTGETQVQKGIEFFRTQAGKMEDEADSH RPSGFEIVFPAMLKEAKILGLDLPYDLPFLKQIIEKREAKLKRIPTDVLYA LPTTLLYSLEGLQEIVDWQKIMKLQSKDGSFLSSPASTAAVFMRTGNKKC LDFLNFVLKKFGNHVPCHYPLDLFERLWAVDTVERLGIDRHFKEEIKEAL DYVYSHWDERGIGWARENPVPDIDDTAMGLRILRLHGYNVSSDVLKTFR DENGEFFCFLGQTQRGVTDMLNVNRCSHVSFPGETIMEEAKLCTERYLRN ALENVDAFDKWAFKKNIRGEVEYALKYPWHKSMPRLEARSYIENYGPDD VWLGKTVYMMPYISNEKYLELAKLDFNKVQSIHQTELQDLRRWWKSSGF TDLNFTRERVTEIYFSPASFIFEPEFSKCREVYTKTSNFTVILDDLYDAHGSL DDLKLFTESVKRWDLSLVDQMPQQMKICFVGFYNTFNDIAKEGRERQGR DVLGYIQNVWKVQLEAYTKEAEWSEAKYVPSFNEYIENASVSIALGTVVL ISALFTGEVLTDEVLSKIDRESRFLQLMGLTGRLVNDTKTYQAERGQGEV ASAIQCYMKDHPKISEEEALQHVYSVMENALEELNREFVNNKIPDIYKRL VFETARIMQLFYMQGDGLTLSHDMEIKEHVKNCLFQPVA GGPPS MFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPA SEQID LCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKP NO.51 TNHKVFGEDTAVLAGDALLSFAFEHIAVATSKTVPSDRTLRVISELGKTIG SQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILGGA TEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYP KLMGLEKAKEFAAELATRAKEELSSFDQIKAAPLLGLADYIAFRQN P450 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVIR SEQID YLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKA NO.52 HNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRL TLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAY DENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEP LDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLV DPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLE KGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQR ACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKA KSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTAR DLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQF VDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAE NIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSL QFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASY QEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVS VEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVL AKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVS VVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLI MVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEE LENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYI CGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDV WAG LOV2 AATLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPET SEQID DRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ NO.53 YFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL BphP1 MASVAGHASGSPAFGTADLSNCEREEIHLAGSIQPHGALLVVSEPDHRIIQ SEQID ASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGN NO.54 PSTEYDGLMHRPPEGGLIIELERAGPPIDLSGTLAPALERIRTAGSLRALCD DTALLFQQCTGYDRVMVYRFDEQGHGEVFSERHVPGLESYFGNRYPSSDI PQMARRLYERQRVRVLVDVSYQPVPLEPRLSPLTGRDLDMSGCFLRSMSP IHLQYLKNMGVRATLVVSLVVGGKLWGLVACHHYLPRFMHFELRAICEL LAEAIATRITALESFAQSQSELFVQRLEQRMIEAITREGDWRAAIFDTSQSIL QPLHAAGCALVYEDQIRTIGDVPSTQDVREIAGWLDRQPRAAVTSTASLG LDVPELAHLTRMASGVVAAPISDHRGEFLMWFRPERVHTVTWGGDPKKP FTMGDTPADLSPRRSFAKWHQVVEGTSDPWTAADLAAARTIGQTVADIV LQFRAVRTLIAREQYEQFSSQVHASMQPVLITDAEGRILLMNDSFRDMLP AGSPSAVHLDDLAGFFVESNDFLRNVAELIDHGRGWRGEVLLRGAGNRP LPLAVRADPVTRTEDQSLGFVLIFSDATDRRTADAARTRFQEGILASARPG VRLDSKSDLLHEKLLSALVENAQLAALEITYGVETGRIAELLEGVRQSML RTAEVLGHLVQHAARTAGSDSSSNGSQNKKEFDSAGSAGSAGTS TC-PTP MGMPTTIEREFEELDTQRRWQPLYLEIRNESHDYPHRVAKFPENRNRNRY SEQID RDVSPYDHSRVKLQNAENDYINASLVDIEEAQRSYILTQGPLPNTCCHFW No.55 LMVWQQKTKAVVMLNRIVEKESVKCAQYWPTDDQEMLFKETGFSVKLL SEDVKSYYTVHLLQLENINSGETRTISHFHYTTWPDFGVPESPASFLNFLFK VRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGDDINIKQVLL NMRKYRMGLIQTPDQLRFSYMAIIEGAKCIKGDSSIQKRWKELS PTP1B.sub.1-435 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDV SEQID SPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMV No.56 WEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISED IKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLL EMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEP PPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEKGSP LNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDEDHAL SYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT SacB MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISHITRHD SEQID MLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVAN No.57 YHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDK FDANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVN VSASDSSLNINGVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHTLRDP HYVEDKGHKYLVFEANTGTEDGYQGEESLFNKAYYGKSTSFFRQESQKL LQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDEIERANVF KMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGL VLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAP SFLLNIKGKKTSVVKDSILEQGQLTVNK GalK MSLKEKTQSLFANAFGYPATHTIQAPGRVNLIGEHTDYNDGFVLPCAIDY SEQID QTVISCAPRDDRKVRVMAADYENQLDEFSLDAPIVAHENYQWANYVRG No.58 VVKHLQLRNNSFGGVDMVISGNVPQGAGLSSSASLEVAVGTVLQQLYHL PLDGAQIALNGQEAENQFVGCNCGIMDQLISALGKKDHALLIDCRSLGTK AVSMPKGVAVVIINSNFKRTLVGSEYNTRREQCETGARFFQQPALRDVTIE EFNAVAHELDPIVAKRVRHILTENARTVEAASALEQGDLKRMGELMAES HASMRDDFEITVPQIDTLVEIVKAVIGDKGGVRMTGGGFGGCIVALIPEEL VPAVQQAVAEQYEAKTGIKETFYVCKPSQGAGQC GHS MAQISESVSPSTDLKSTESSITSNRHGNMWEDDRIQSLNSPYGAPAYQERS SEQID EKLIEEIKLLFLSDMDDSCNDSDRDLIKRLEIVDTVECLGIDRHFQPEIKLAL No.59 DYVYRCWNERGIGEGSRDSLKKDLNATALGFRALRLHRYNVSSGVLENF RDDNGQFFCGSTVEEEGAEAYNKHVRCMLSLSRASNILFPGEKVMEEAK AFTTNYLKKVLAGREATHVDESLLGEVKYALEFPWHCSVQRWEARSFIEI FGQIDSELKSNLSKKMLELAKLDFNILQCTHQKELQIISRWFADSSIASLNF YRKCYVEFYFWMAAAISEPEFSGSRVAFTKIAILMTMLDDLYDTHGTLDQ LKIFTEGVRRWDVSLVEGLPDFMKIAFEFWLKTSNELIAEAVKAQGQDMA AYIRKNAWERYLEAYLQDAEWIATGHVPTFDEYLNNGTPNTGMCVLNLI PLLLMGEHLPIDILEQIFLPSRFHHLIELASRLVDDARDFQAEKDHGDLSCIE CYLKDHPESTVEDALNHVNGLLGNCLLEMNWKFLKKQDSVPLSCKKYSF HVLARSIQFMYNQGDGFSISNKVIKDQVQKVLIVPVPI* ADS MALTEEKPIRPIANFPPSIWGDQFLIYEKQVEQGVEQIVNDLKKEVRQLLK SEQID EALDIPMKHANLLKLIDEIQRLGIPYHFEREIDHALQCIYETYGDNWNGDR No.60 SSLWFRLMRKQGYYVTCDVFNNYKDKNGAFKQSLANDVEGLLELYEAT SMRVPGEIILEDALGFTRSRLSIMTKDAFSTNPALFTEIQRALKQPLWKRLP RIEAAQYIPFYQQQDSHNKTLLKLAKLEFNLLQSLHKEELSHVCKWWKAF DIKKNAPCLRDRIVECYFWGLGSGYEPQYSRARVFFTKAVAVITLIDDTYD AYGTYEELKIFTEAVERWSITCLDTLPEYMKPIYKLFMDTYTEMEEFLAKE GRTDLFNCGKEFVKEFVRNLMVEAKWANEGHIPTTEEHDPVVIITGGANL LTTTCYLGMSDIFTKESVEWAVSAPPLFRYSGILGRRLNDLMTHKAEQER KHSSSSLESYMKEYNVNEEYAQTLIYKEVEDVWKDINREYLTTKNIPRPLL MAVIYLCQFLEVQYAGKDNFTRMGDEYKHLIKSLLVYPMSI* TXS MSSSTGTSKVVSETSSTIVDDIPRLSANYHGDLWHHNVIQTLETPFRESSTY SEQID QERADELVVKIKDMFNALGDGDISPSAYDTAWVARLATISSDGSEKPRFP No.61 QALNWVFNNQLQDGSWGIESHFSLCDRLLNTTNSVIALSVWKTGHSQVQ QGAEFIAENLRLLNEEDELSPDFQIIFPALLQKAKALGINLPYDLPFIKYLST TREARLTDVSAAADNIPANMLNALEGLEEVIDWNKIMRFQSKDGSFLSSP ASTACVLMNTGDEKCFTFLNNLLDKFGGCVPCMYSIDLLERLSLVDNIEH LGIGRHFKQEIKGALDYVYRHWSERGIGWGRDSLVPDLNTTALGLRTLR MHGYNVSSDVLNNFKDENGRFFSSAGQTHVELRSVVNLFRASDLAFPDE RAMDDARKFAEPYLREALATKISTNTKLFKEIEYVVEYPWHMSIPRLEAR SYIDSYDDNYVWQRKTLYRMPSLSNSKCLELAKLDFNIVQSLHQEELKLL TRWWKESGMADINFTRHRVAEVYFSSATFEPEYSATRIAFTKIGCLQVLFD DMADIFATLDELKSFTEGVKRWDTSLLHEIPECMQTCFKVWFKLMEEVN NDVVKVQGRDMLAHIRKPWELYFNCYVQEREWLEAGYIPTFEEYLKTYA ISVGLGPCTLQPILLMGELVKDDVVEKVHYPSNMFELVSLSWRLTNDTKT YQAEKARGQQASGIACYMKDNPGATEEDAIKHICRVVDRALKEASFEYF KPSNDIPMGCKSFIFNLRLCVQIFYKFIDGYGIANEEIKDYIRKVYIDPIQV* TC-PTP MPTTIEREFEELDTQRRWQPLYLEIRNESHDYPHRVAKFPENRNRNRYRD SEQID VSPYDHSRVKLQNAENDYINASLVDIEEAQRSYILTQGPLPNTCCHFWLM No.62 VWQQKTKAVVMLNRIVEKESVKCAQYWPTDDQEMLFKETGFSVKLLSE DVKSYYTVHLLQLENINSGETRTISHFHYTTWPDFGVPESPASFLNFLFKV RESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGDDINIKQVLLN MRKYRMGLIQTPDQLRFSYMAIIEGAKCIKGDSSIQKRWKELSKEDLSPAF DHSPNKIMTEKYNGNR PTPN5 MSSGVDLGTENLYFQSMSRVLQAEELHEKALDPFLLQAEFFEIPMNFVDP SEQID KEYDIPGLVRKNRYKTILPNPHSRVCLTSPDPDDPLSSYINANYIRGYGGEE No.63 KVYIATQGPIVSTVADFWRMVWQEHTPIIVMITNIEEMNEKCTEYWPEEQ VAYDGVEITVQKVIHTEDYRLRLISLKSGTEERGLKHYWFTSWPDQKTPD RAPPLLHLVREVEEAAQQEGPHCAPIIVHCSAGIGRTGCFIATSICCQQLRQ EGVVDILKTTCQLRQDRGGMIQTCEQYQFVHHVMSLYEKQLSHQS* PTPN6 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQV SEQID THIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVVQDRDGTIIHLKYPL No.64 NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSV LSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGI EEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEF ESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGS DYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIV MTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQ VSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHA GPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGM VQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNA HAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK* PTPN11 MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGA SEQID VTHIKIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELK No.65 YPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVL SVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYK KNPMVETLGTVLQLKQPLNTTRINAAEIESRVRELSKLAETTDKVKQGFW EEFETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDP NEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQ ENSRVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHD YTLRELKLSKVGQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVHH KQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQMV RSQRSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIKY SLADQTSGDQSPLPPCTPTPPCAEMREDSARVYENVGLMQQQKSFR* PTN12 MEQVEILRKFIQRVQAMKSPDHNGEDNFARDFMRLRRLSTKYRTEKIYPT SEQID ATGEKEENVKKNRYKDILPFDHSRVKLTLKTPSQDSDYINANFIKGVYGP No.66 KAYVATQGPLANTVIDFWRMVWEYNVVIIVMACREFEMGRKKCERYWP LYGEDPITFAPFKISCEDEQARTDYFIRTLLLEFQNESRRLYQFHYVNWPD HDVPSSFDSILDMISLMRKYQEHEDVPICIHCSAGCGRTGAICAIDYTWNL LKAGKIPEEFNVFNLIQEMRTQRHSAVQTKEQYELVHRAIAQLFEKQLQL YEIHGAQKIADGVNEINTENMVSSIEPEKQDSPPPKPPRTRSCLVEGDAKEE ILQPPEPHPVPPILTPSPPSAFPTVTTVWQDNDRYHPKPVLQWFHQNNIQQT STETIVNQQNFQGKMNQQLNR PTPN22 MDQREILQKFLDEAQSKKITKEEFANEFLKLKRQSTKYKADKTYPTTVAE SEQID KPKNIKKNRYKDILPYDYSRVELSLITSDEDSSYINANFIKGVYGPKAYIAT No.67 QGPLSTTLLDFWRMIWEYSVLIIVMACMEYEMGKKKCERYWAEPGEMQ LEFGPFSVSCEAEKRKSDYIIRTLKVKFNSETRTTYQFHYKNWPDHDVPSSI DPILELIWDVRCYQEDDSVPICIHCSAGCGRTGVICAIDYTWMLLKDGIIPE NFSVFSLIREMRTQRPSLVQTQEQYELVYNAVLELFKRQMDVIRD sfGFP MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTT SEQID GKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIS No.68 FKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHN VYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK* mClover MHHHHHHVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKL SEQID TLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGY No.69 VQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSHQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

TABLE-US-00006 TABLE4 ExemplaryTerminators. SEQ IDNo. Name DNASequence # T7 ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGG SEQID CCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTT No.70 CCTTTCGGGCTTTGTTAGCAG rrnB GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAA SEQID T1/T2 CGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGT No.71 CCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTA GTGTGGGGTCACCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACG AAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAA CGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGC AACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAA ATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCT TrrnB TGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAA SEQID GTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGG No.72 GAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT

TABLE-US-00007 TABLE5 ExemplaryDNASequences(includestruncations): SEQ ID Comp. Organism DNASequence No.# Src H.sapiens ATGGGCTCCAAGCCGCAGACTCAGGGCCTGGCCAAGGATGCCTGGGA SEQ GATCCCTCGGGAGTCGCTGCGGCTGGAGGTCAAGCTGGGCCAGGGCT ID GCTTTGGCGAGGTGTGGATGGGGACCTGGAACGGTACCACCAGGGTG No.73 GCCATCAAAACCCTGAAGCCTGGCACGATGTCTCCAGAGGCCTTCCTG CAGGAGGCCCAGGTCATGAAGAAGCTGAGGCATGAGAAGCTGGTGCA GTTGTATGCTGTGGTTTCAGAGGAGCCCATTTACATCGTCACGGAGTA CATGAGCAAGGGGAGTTTGCTGGACTTTCTCAAGGGGGAGACAGGCA AGTACCTGCGGCTGCCTCAGCTGGTGGACATGGCTGCTCAGATCGCCT CAGGCATGGCGTACGTGGAGCGGATGAACTACGTCCACCGGGACCTTC GTGCAGCCAACATCCTGGTGGGAGAGAACCTGGTGTGCAAAGTGGCC GACTTTGGGCTGGCTCGGCTCATTGAAGACAATGAGTACACGGCGCGG CAAGGTGCCAAATTCCCCATCAAGTGGACGGCTCCAGAAGCTGCCCTC TATGGCCGCTTCACCATCAAGTCGGACGTGTGGTCCTTCGGGATCCTG CTGACTGAGCTCACCACAAAGGGACGGGTGCCCTACCCTGGGATGGTG AACCGCGAGGTGCTGGACCAGGTGGAGCGGGGCTACCGGATGCCCTG CCCGCCGGAGTGTCCCGAGTCCCTGCACGACCTCATGTGCCAGTGCTG GCGGAAGGAGCCTGAGGAGCGGCCCACCTTCGAGTACCTGCAGGCCT TCCTGGAGGACTACTTCACGTCCACCGAGCCCCAGTACCAGCCCGGGG AGAACCTCTAA CDC37 H.sapiens ATGGTGGACTACAGCGTGTGGGACCACATTGAGGTGTCTGATGATGAA SEQ GACGAGACGCACCCCAACATCGACACGGCCAGTCTCTTCCGCTGGCGG ID CATCAGGCCCGGGTGGAACGCATGGAGCAGTTCCAGAAGGAGAAGGA No.74 GGAACTGGACAGGGGCTGCCGCGAGTGCAAGCGCAAGGTGGCCGAGT GCCAGAGGAAACTGAAGGAGCTGGAGGTGGCCGAGGGCGGCAAGGC AGAGCTGGAGCGCCTGCAGGCCGAGGCACAGCAGCTGCGCAAGGAGG AGCGGAGCTGGGAGCAGAAGCTGGAGGAGATGCGCAAGAAGGAGAA GAGCATGCCCTGGAACGTGGACACGCTCAGCAAAGACGGCTTCAGCA AGAGCATGGTAAATACCAAGCCCGAGAAGACGGAGGAGGACTCAGAG GAGGTGAGGGAGCAGAAACACAAGACCTTCGTGGAAAAATACGAGAA ACAGATCAAGCACTTTGGCATGCTTCGCCGCTGGGATGACAGCCAAAA GTACCTGTCAGACAACGTCCACCTGGTGTGCGAGGAGACAGCCAATTA CCTGGTCATTTGGTGCATTGACCTAGAGGTGGAGGAGAAATGTGCACT CATGGAGCAGGTGGCCCACCAGACAATCGTCATGCAATTTATCCTGGA GCTGGCCAAGAGCCTAAAGGTGGACCCCCGGGCCTGCTTCCGGCAGTT CTTCACTAAGATTAAGACAGCCGATCGCCAGTACATGGAGGGCTTCAA CGACGAGCTGGAAGCCTTCAAGGAGCGTGTGCGGGGCCGTGCCAAGC TGCGCATCGAGAAGGCCATGAAGGAGTACGAGGAGGAGGAGCGCAA GAAGCGGCTCGGCCCCGGCGGCCTGGACCCCGTCGAGGTCTACGAGTC CCTCCCTGAGGAACTCCAGAAGTGCTTCGATGTGAAGGACGTGCAGAT GCTGCAGGACGCCATCAGCAAGATGGACCCCACCGACGCAAAGTACC ACATGCAGCGCTGCATTGACTCTGGCCTCTGGGTCCCCAACTCTAAGG CCAGCGAGGCCAAGGAGGGAGAGGAGGCAGGTCCTGGGGACCCATTA CTGGAAGCTGTTCCCAAGACGGGCGATGAGAAGGATGTCAGTGTGTA A PTP1B.sub.1-435 H.sapiens ATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAGTCCGGGAGCTG SEQ GGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATG ID TAGAGTGGCCAAGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAG No.75 ACGTCAGTCCCTTTGACCATAGTCGGATTAAACTACATCAAGAAGATA ATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGG AGTTACATTCTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTT GGGAGATGGTGTGGGAGCAGAAAAGCAGGGGTGTCGTCATGCTCAAC AGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACA AAAAGAAGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAA CATTGATCTCTGAAGATATCAAGTCATATTATACAGTGCGACAGCTAG AATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCC ACTATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCAT TCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGCCCGG AGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTG GAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGA AAGACCCTTCTTCCGTTGATATCAAGAAAGTGCTGTTAGAAATGAGGA AGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTTCTCCT ACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCG TGCAGGATCAGTGGAAGGAGCTTTCCCACGAGGACCTGGAGCCCCCA CCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAATCCTGGAG CCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTG AAGGAAGAGACCCAGGAGGATAAAGACTGCCCCATCAAGGAAGAAA AAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAAAGCATGAGT CAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGG TGCCCAGGCTGCCTCCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGA AGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTTCCTGGTCAACA TGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGT TCCTGTTCAACAGCAACACATAG LuxAB Bacterial ATGAAATTTGGAAACTTTTTGCTTACATACCAACCTCCCCAATTTTCCC SEQ AAACAGAGGTAATGAAACGTTTGGTTAAATTAGGTCGCATCTCTGAGG ID AGTGTGGTTTTGATACCGTATGGTTACTGGAGCATCATTTCACGGAGTT No.76 TGGTTTGCTTGGTAACCCTTATGTCGCTGCTGCATATTTACTTGGCGCG ACTAAAAAATTGAATGTAGGAACTGCCGCTATTGTTCTTCCCACAGCC CATCCAGTACGCCAACTTGAAGATGTGAATTTATTGGATCAAATGTCA AAAGGACGATTTCGGTTTGGTATTTGCCGAGGGCTTTACAACAAGGAC TTTCGCGTATTCGGCACAGATATGAATAACAGTCGCGCCTTAGCGGAA TGCTGGTACGGGCTGATAAAGAATGGCATGACAGAGGGATATATGGA AGCTGATAATGAACATATCAAGTTCCATAAGGTAAAAGTAAACCCCGC GGCGTATAGCAGAGGTGGCGCACCGGTTTATGTGGTGGCTGAATCAGC TTCGACGACTGAGTGGGCTGCTCAATTTGGCCTACCGATGATATTAAG TTGGATTATAAATACTAACGAAAAGAAAGCACAACTTGAGCTTTATAA TGAAGTGGCTCAAGAATATGGGCACGATATTCATAATATCGACCATTG CTTATCATATATAACATCTGTAGATCATGACTCAATTAAAGCGAAAGA GATTTGCCGGAAATTTCTGGGGCATTGGTATGATTCTTATGTGAATGCT ACGACTATTTTTGATGATTCAGACCAAACAAGAGGTTATGATTTCAAT AAAGGGCAGTGGCGTGACTTTGTATTAAAAGGACATAAAGATACTAA TCGCCGTATTGATTACAGTTACGAAATCAATCCCGTGGGAACGCCGCA GGAATGTATTGACATAATTCAAAAAGACATTGATGCTACAGGAATATC AAATATTTGTTGTGGATTTGAAGCTAATGGAACAGTAGACGAAATTAT TGCTTCCATGAAGCTCTTCCAGTCTGATGTCATGCCATTTCTTAAAGAA AAACAACGTTCGCTATTATATTATTAA LuxB V.fischeri ATGAGCAAATTTGGATTGTTCTTCCTTAACTTCATCAATTCAACAACTG SEQ TTCAAGAACAGAGTATAGTTCGCATGCAGGAAATAACGGAGTATGTTG ID ATAAGTTGAATTTTGAACAGATTTTAGTGTATGAAAATCATTTTTCAGA No.77 TAATGGTGTTGTCGGCGCTCCTCTGACTGTTTCTGGTTTTCTGCTCGGT TTAACAGAGAAAATTAAAATTGGTTCATTAAATCACATCATTACAACT CATCATCCTGTCCGCATAGCGGAGGAAGCTTGCTTATTGGATCAGTTA AGTGAAGGGAGATTTATTTTAGGGTTTAGTGATTGCGAAAAAAAAGAT GAAATGCATTTTTTTAATCGCCCGGTTGAATATCAACAGCAACTATTTG AAGAGTGTTATGAAATCATTAACGATGCTTTAACAACAGGCTATTGTA ATCCAGATAACGATTTTTATAGCTTCCCTAAAATATCTGTAAATCCCCA TGCTTATACGCCAGGCGGACCTCGGAAATATGTAACAGCAACCAGTCA TCATATTGTTGAGTGGGCGGCCAAAAAAGGTATTCCTCTCATCTTTAA GTGGGATGATTCTAATGATGTTAGATATGAATATGCTGAAAGATATAA AGCCGTTGCGGATAAATATGACGTTGACCTATCAGAGATAGACCATCA GTTAATGATATTAGTTAACTATAACGAAGATAGTAATAAAGCTAAACA AGAAACGCGTGCATTTATTAGTGATTATGTTCTTGAAATGCACCCTAA TGAAAATTTCGAAAATAAACTTGAAGAAATAATTGCAGAAAACGCTG TCGGAAATTATACGGAGTGTATAACTGCGGCTAAGTTGGCAATTGAAA AGTGTGGTGCGAAAAGTGTATTGCTGTCCTTTGAACCAATGAATGATT TGATGAGCCAAAAAAATGTAATCAATATTGTTGATGATAATATTAAGA AGTACCACACGGAATATACCTAA RpoZ Escherichia ATGGCACGCGTAACTGTTCAGGACGCTGTAGAGAAAATTGGTAACCGT SEQ coli TTTGACCTGGTACTGGTCGCCGCGCGTCGCGCTCGTCAGATGCAGGTA ID GGCGGAAAGGATCCGCTGGTACCGGAAGAAAACGATAAAACCACTGT No.78 AATCGCGCTGCGCGAAATCGAAGAAGGTCTGATCAACAACCAGATCC TCGACGTTCGCGAACGCCAGGAACAGCAAGAGCAGGAAGCCGCTGAA TTACAAGCCGTTACCGCTATTGCTGAAGGTCGTCGTTAA cI Lambda ATGAGTATCAGCAGCAGGGTAAAAAGCAAAAGAATTCAGCTTGGACT SEQ bacterio- TAACCAGGCTGAACTTGCTCAAAAGGTGGGGACTACCCAGCAGTCTAT ID phage AGAGCAGCTCGAAAACGGTAAAACTAAGCGACCACGCTTTTTACCAG No.79 AACTTGCGTCAGCTCTTGGCGTAAGTGTTGACTGGCTGCTCAATGGCA CCTCTGATTCGAATGTTAGATTTGTTGGGCACGTTGAGCCCAAAGGGA AATATCCATTGATTAGCATGGTTAGAGCTCGTTCGTGGTGTGAAGCTT GTGAACCCTACGATATCAAGGACATTGATGAATGGTATGACAGTGACG TTAACTTATTAGGCAATGGATTCTGGCTGAAGGTTGAAGGTGATTCCA TGACCTCACCTGTAGGTCAAAGCATCCCTGAAGGTCATATGGTGTTAG TAGATACTGGACGGGAGCCAGTGAATGGAAGCCTTGTTGTAGCCAAA CTGACTGACGCGAACGAAGCAACATTCAAGAAACTGGTCATAGATGG CGGTCAGAAGTACCTGAAAGGCCTGAATCCTTCATGGCCTATGACTCC TATCAACGGAAACTGCAAGATTATCGGTGTTGTCGTGGAAGCGAGGGT AAAATTCGTAGACTAA SH2 Rous ATGTGGTATTTTGGGAAGATCACTCGTCGGGAGTCCGAGCGGCTGCTG SEQ sarcoma CTCAACCCCGAAAACCCCCGGGGAACCTTCTTGGTCCGGGAGAGCGA ID virus GACGGTAAAAGGTGCCTATGCCCTCTCCGTTTCTGACTTTGACAACGC No.80 CAAGGGGCTCAATGTGAAACACTACCTGATCCGCAAGCTGGACAGCG GCGGCTTCTACATCACCTCACGCACACAGTTCAGCAGCCTGCAGCAGC TGGTGGCCTACTACTCCAAACATGCTGATGGCTTGTGCCACCGCCTGA CCAACGTCTGCTAA MBP E.coli ATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAA SEQ AGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATA ID CCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAA No.81 TTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGG GCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAA ATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGG GATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTT GAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCA AAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAA AGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTG GCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGG CAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAG CGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATG CAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAA CAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACC AGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAA CCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCC AGTCCGAACAAAGAGCTGGCGAAAGAGTTCCTCGAAAACTATCTGCT GACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTG CCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGT ATTGCCGCCACCATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAA CATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATC AACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGC GCAGACTCGTATCACCAAGTAA p130cas H.sapiens TGGATGGAGGACTATGACTACGTCCACCTACAGGGG SEQ (or ID Kras) No.82 substrate MidT Hamster GAACCGCAGTATGAAGAAATTCCGATTTATCTG SEQ substrate polyoma ID virus No.83 EGFR H.sapiens CCGCAGCGCTATCTGGTGATTCAGGGCGAT SEQ substrate ID No.84 ShcA H.sapiens GATCATCAGTATTATAACGATTTTCCGGGC SEQ substrate ID No.85 MBIS S. ATGTCATTACCGTTCTTAACTTCTGCACCGGGAAAGGTTATTATTTTTG SEQ cerevisiae GTGAACACTCTGCTGTGTACAACAAGCCTGCCGTCGCTGCTAGTGTGT ID (from CTGCGTTGAGAACCTACCTGCTAATAAGCGAGTCATCTGCACCAGATA No.86 pMBIS CTATTGAATTGGACTTCCCGGACATTAGCTTTAATCATAAGTGGTCCAT Addgene#: CAATGATTTCAATGCCATCACCGAGGATCAAGTAAACTCCCAAAAATT 17817 GGCCAAGGCTCAACAAGCCACCGATGGCTTGTCTCAGGAACTCGTTAG TCTTTTGGATCCGTTGTTAGCTCAACTATCCGAATCCTTCCACTACCAT GCAGCGTTTTGTTTCCTGTATATGTTTGTTTGCCTATGCCCCCATGCCA AGAATATTAAGTTTTCTTTAAAGTCTACTTTACCCATCGGTGCTGGGTT GGGCTCAAGCGCCTCTATTTCTGTATCACTGGCCTTAGCTATGGCCTAC TTGGGGGGGTTAATAGGATCTAATGACTTGGAAAAGCTGTCAGAAAA CGATAAGCATATAGTGAATCAATGGGCCTTCATAGGTGAAAAGTGTAT TCACGGTACCCCTTCAGGAATAGATAACGCTGTGGCCACTTATGGTAA TGCCCTGCTATTTGAAAAAGACTCACATAATGGAACAATAAACACAAA CAATTTTAAGTTCTTAGATGATTTCCCAGCCATTCCAATGATCCTAACC TATACTAGAATTCCAAGGTCTACAAAAGATCTTGTTGCTCGCGTTCGT GTGTTGGTCACCGAGAAATTTCCTGAAGTTATGAAGCCAATTCTAGAT GCCATGGGTGAATGTGCCCTACAAGGCTTAGAGATCATGACTAAGTTA AGTAAATGTAAAGGCACCGATGACGAGGCTGTAGAAACTAATAATGA ACTGTATGAACAACTATTGGAATTGATAAGAATAAATCATGGACTGCT TGTCTCAATCGGTGTTTCTCATCCTGGATTAGAACTTATTAAAAATCTG AGCGATGATTTGAGAATTGGCTCCACAAAACTTACCGGTGCTGGTGGC GGCGGTTGCTCTTTGACTTTGTTACGAAGAGACATTACTCAAGAGCAA ATTGACAGCTTCAAAAAGAAATTGCAAGATGATTTTAGTTACGAGACA TTTGAAACAGACTTGGGTGGGACTGGCTGCTGTTTGTTAAGCGCAAAA AATTTGAATAAAGATCTTAAAATCAAATCCCTAGTATTCCAATTATTTG AAAATAAAACTACCACAAAGCAACAAATTGACGATCTATTATTGCCAG GAAACACGAATTTNCCATGGACTTCATAGGAGGCAGATCAAATGTCA GAGTTGAGAGCCTTCAGTGCCCCAGGGAAAGCGTTACTAGCTGGTGGA TATTTAGTTTTAGATACAAAATATGAAGCATTTGTAGTCGGATTATCG GCAAGAATGCATGCTGTAGCCCATCCTTACGGTTCATTGCAAGGGTCT GATAAGTTTGAAGTGCGTGTGAAAAGTAAACAATTTAAAGATGGGGA GTGGCTGTACCATATAAGTCCTAAAAGTGGCTTCATTCCTGTTTCGATA GGCGGATCTAAGAACCCTTTCATTGAAAAAGTTATCGCTAACGTATTT AGCTACTTTAAACCTAACATGGACGACTACTGCAATAGAAACTTGTTC GTTATTGATATTTTCTCTGATGATGCCTACCATTCTCAGGAGGATAGCG TTACCGAACATCGTGGCAACAGAAGATTGAGTTTTCATTCGCACAGAA TTGAAGAAGTTCCCAAAACAGGGCTGGGCTCCTCGGCAGGTTTAGTCA CAGTTTTAACTACAGCTTTGGCCTCCTTTTTTGTATCGGACCTGGAAAA TAATGTAGACAAATATAGAGAAGTTATTCATAATTTAGCACAAGTTGC TCATTGTCAAGCTCAGGGTAAAATTGGAAGCGGGTTTGATGTAGCGGC GGCAGCATATGGATCTATCAGATATAGAAGATTCCCACCCGCATTAAT CTCTAATTTGCCAGATATTGGAAGTGCTACTTACGGCAGTAAACTGGC GCATTTGGTTGATGAAGAAGACTGGAATATTACGATTAAAAGTAACCA TTTACCTTCGGGATTAACTTTATGGATGGGCGATATTAAGAATGGTTC AGAAACAGTAAAACTGGTCCAGAAGGTAAAAAATTGGTATGATTCGC ATATGCCAGAAAGCTTGAAAATATATACAGAACTCGATCATGCAAATT CTAGATTTATGGATGGACTATCTAAACTAGATCGCTTACACGAGACTC ATGACGATTACAGCGATCAGATATTTGAGTCTCTTGAGAGGAATGACT GTACCTGTCAAAAGTATCCTGAAATCACAGAAGTTAGAGATGCAGTTG CCACAATTAGACGTTCCTTTAGAAAAATAACTAAAGAATCTGGTGCCG ATATCGAACCTCCCGTACAAACTAGCTTATTGGATGATTGCCAGACCT TAAAAGGAGTTCTTACTTGCTTAATACCTGGTGCTGGTGGTTATGACG CCATTGCAGTGATTACTAAGCAAGATGTTGATCTTAGGGCTCAAACCG CTAATGACAAAAGATTTTCTAAGGTTCAATGGCTGGATGTAACTCAGG CTGACTGGGGTGTTAGGAAAGAAAAAGATCCGGAAACTTATCTTGATA AATAGGAGGTAATACTCATGACCGTTTACACAGCATCCGTTACCGCAC CCGTCAACATCGCAACCCTTAAGTATTGGGGGAAAAGGGACACGAAG TTGAATCTGCCCACCAATTCGTCCATATCAGTGACTTTATCGCAAGATG ACCTCAGAACGTTGACCTCTGCGGCTACTGCACCTGAGTTTGAACGCG ACACTTTGTGGTTAAATGGAGAACCACACAGCATCGACAATGAAAGA ACTCAAAATTGTCTGCGCGACCTACGCCAATTAAGAAAGGAAATGGA ATCGAAGGACGCCTCATTGCCCACATTATCTCAATGGAAACTCCACAT TGTCTCCGAAAATAACTTTCCTACAGCAGCTGGTTTAGCTTCCTCCGCT GCTGGCTTTGCTGCATTGGTCTCTGCAATTGCTAAGTTATACCAATTAC CACAGTCAACTTCAGAAATATCTAGAATAGCAAGAAAGGGGTCTGGTT CAGCTTGTAGATCGTTGTTTGGCGGATACGTGGCCTGGGAAATGGGAA AAGCTGAAGATGGTCATGATTCCATGGCAGTACAAATCGCAGACAGCT CTGACTGGCCTCAGATGAAAGCTTGTGTCCTAGTTGTCAGCGATATTA AAAAGGATGTGAGTTCCACTCAGGGTATGCAATTGACCGTGGCAACCT CCGAACTATTTAAAGAAAGAATTGAACATGTCGTACCAAAGAGATTTG AAGTCATGCGTAAAGCCATTGTTGAAAAAGATTTCGCCACCTTTGCAA AGGAAACAATGATGGATTCCAACTCTTTCCATGCCACATGTTTGGACT CTTTCCCTCCAATATTCTACATGAATGACACTTCCAAGCGTATCATCAG TTGGTGCCACACCATTAATCAGTTTTACGGAGAAACAATCGTTGCATA CACGTTTGATGCAGGTCCAAATGCTGTGTTGTACTACTTAGCTGAAAA TGAGTCGAAACTCTTTGCATTTATCTATAAATTGTTTGGCTCTGTTCCT GGATGGGACAAGAAATTTACTACTGAGCAGCTTGAGGCTTTCAACCAT CAATTTGAATCATCTAACTTTACTGCACGTGAATTGGATCTTGAGTTGC AAAAGGATGTTGCCAGAGTGATTTTAACTCAAGTCGGTTCAGGCCCAC AAGAAACAAACGAATCTTTGATTGACGCAAAGACTGGTCTACCAAAG GAATAACTGCAGCCCGGGAGGAGGATTACTATATGCAAACGGAACAC GTCATTTTATTGAATGCACAGGGAGTTCCCACGGGTACGCTGGAAAAG TATGCCGCACACACGGCAGACACCCGCTTACATCTCGCGTTCTCCAGT TGGCTGTTTAATGCCAAAGGACAATTATTAGTTACCCGCCGCGCACTG AGCAAAAAAGCATGGCCTGGCGTGTGGACTAACTCGGTTTGTGGGCAC CCACAACTGGGAGAAAGCAACGAAGACGCAGTGATCCGCCGTTGCCG TTATGAGCTTGGCGTGGAAATTACGCCTCCTGAATCTATCTATCCTGAC TTTCGCTACCGCGCCACCGATCCGAGTGGCATTGTGGAAAATGAAGTG TGTCCGGTATTTGCCGCACGCACCACTAGTGCGTTACAGATCAATGAT GATGAAGTGATGGATTATCAATGGTGTGATTTAGCAGATGTATTACAC GGTATTGATGCCACGCCGTGGGCGTTCAGTCCGTGGATGGTGATGCAG GCGACAAATCGCGAAGCCAGAAAACGATTATCTGCATTTACCCAGCTT AAATAACCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGA GGAGGAATGAGTAATGGACTTTCCGCAGCAACTCGAAGCCTGCGTTAA GCAGGCCAACCAGGCGCTGAGCCGTTTTATCGCCCCACTGCCCTTTCA GAACACTCCCGTGGTCGAAACCATGCAGTATGGCGCATTATTAGGTGG TAAGCGCCTGCGACCTTTCCTGGTTTATGCCACCGGTCATATGTTCGGC GTTAGCACAAACACGCTGGACGCACCCGCTGCCGCCGTTGAGTGTATC CACGCTTACTCATTAATTCATGATGATTTACCGGCAATGGATGATGAC GATCTGCGTCGCGGTTTGCCAACCTGCCATGTGAAGTTTGGCGAAGCA AACGCGATTCTCGCTGGCGACGCTTTACAAACGCTGGCGTTCTCGATT TTAAGCGATGCCGATATGCCGGAAGTGTCGGACCGCGACAGAATTTCG ATGATTTCTGAACTGGCGAGCGCCAGTGGTATTGCCGGAATGTGCGGT GGTCAGGCATTAGATTTAGACGCGGAAGGCAAACACGTACCTCTGGA CGCGCTTGAGCGTATTCATCGTCATAAAACCGGCGCATTGATTCGCGC CGCCGTTCGCCTTGGTGCATTAAGCGCCGGAGATAAAGGACGTCGTGC TCTGCCGGTACTCGACAAGTATGCAGAGAGCATCGGCCTTGCCTTCCA GGTTCAGGATGACATCCTGGATGTGGTGGGAGATACTGCAACGTTGGG AAAACGCCAGGGTGCCGACCAGCAACTTGGTAAAAGTACCTACCCTG CACTTCTGGGTCTTGAGCAAGCCCGGAAGAAAGCCCGGGATCTGATCG ACGATGCCCGTCAGTCGCTGAAACAACTGGCTGAACAGTCACTCGATA CCTCGGCACTGGAAGCGCTAGCGGACTACATCATCCAGCGTAATAAAT AA ADS Artemisia GCCCTGACCGAAGAGAAACCGATCCGCCCGATCGCTAACTTCCCGCCG SEQ annua TCTATCTGGGGTGACCAGTTCCTGATCTACGAAAAGCAGGTTGAGCAG ID GGTGTTGAACAGATCGTAAACGACCTGAAGAAAGAAGTTCGTCAGCT No.87 GCTGAAAGAAGCTCTGGACATCCCGATGAAACACGCTAACCTGTTGAA GCTGATCGACGAGATCCAGCGTCTGGGTATCCCGTACCACTTCGAACG CGAAATCGACCACGCACTGCAGTGCATCTACGAAACCTACGGCGACA ACTGGAACGGCGACCGTTCTTCTCTGTGGTTTCGTCTGATGCGTAAAC AGGGCTACTACGTTACCTGTGACGTTTTTAACAACTACAAGGACAAGA ACGGTGCTTTCAAACAGTCTCTGGCTAACGACGTTGAAGGCCTGCTGG AACTGTACGAAGCGACCTCCATGCGTGTACCGGGTGAAATCATCCTGG AGGACGCGCTGGGTTTCACCCGTTCTCGTCTGTCCATTATGACTAAAG ACGCTTTCTCTACTAACCCGGCTCTGTTCACCGAAATCCAGCGTGCTCT GAAACAGCCGCTGTGGAAACGTCTGCCGCGTATCGAAGCAGCACAGT ACATTCCGTTTTACCAGCAGCAGGACTCTCACAACAAGACCCTGCTGA AACTGGCTAAGCTGGAATTCAACCTGCTGCAGTCTCTGCACAAAGAAG AACTGTCTCACGTTTGTAAGTGGTGGAAGGCATTTGACATCAAGAAAA ACGCGCCGTGCCTGCGTGACCGTATCGTTGAATGTTACTTCTGGGGTCT GGGTTCTGGTTATGAACCACAGTACTCCCGTGCACGTGTGTTCTTCACT AAAGCTGTAGCTGTTATCACCCTGATCGATGACACTTACGATGCTTAC GGCACCTACGAAGAACTGAAGATCTTTACTGAAGCTGTAGAACGCTGG TCTATCACTTGCCTGGACACTCTGCCGGAGTACATGAAACCGATCTAC AAACTGTTCATGGATACCTACACCGAAATGGAGGAATTCCTGGCAAAA GAAGGCCGTACCGACCTGTTCAACTGCGGTAAAGAGTTTGTTAAAGAA TTCGTACGTAACCTGATGGTTGAAGCTAAATGGGCTAACGAAGGCCAT ATCCCGACTACCGAAGAACATGACCCGGTTGTTATCATCACCGGCGGT GCAAACCTGCTGACCACCACTTGCTATCTGGGTATGTCCGACATCTTTA CCAAGGAATCTGTTGAATGGGCTGTTTCTGCACCGCCGCTGTTCCGTTA CTCCGGTATTCTGGGTCGTCGTCTGAACGACCTGATGACCCACAAAGC AGAGCAGGAACGTAAACACTCTTCCTCCTCTCTGGAATCCTACATGAA GGAATATAACGTTAACGAGGAGTACGCACAGACTCTGATCTATAAAG AAGTTGAAGACGTATGGAAAGACATCAACCGTGAATACCTGACTACT AAAAACATCCCGCGCCCGCTGCTGATGGCAGTAATCTACCTGTGCCAG TTCCTGGAAGTACAGTACGCTGGTAAAGATAACTTCACTCGCATGGGC GACGAATACAAACACCTGATCAAATCCCTGCTGGTTTACCCGATGTCC ATCTGA GHS Abies ATGGCTCAAATCAGCGAATCAGTGTCTCCAAGCACCGACCTTAAAAGC SEQ grandis ACGGAATCTTCTATTACCAGCAACCGCCACGGTAACATGTGGGAAGAT ID GACCGCATTCAGAGCTTAAACAGCCCATATGGCGCACCCGCTTATCAG No.88 GAACGTAGCGAAAAATTGATTGAAGAAATTAAGCTCCTGTTTCTGTCC GATATGGACGATAGTTGCAATGATTCGGATCGCGACTTGATCAAACGC CTGGAGATCGTAGATACGGTTGAGTGTCTGGGCATTGATCGTCATTTC CAACCTGAAATTAAGCTGGCGCTGGATTACGTGTACCGTTGCTGGAAT GAGCGTGGCATCGGAGAAGGTAGCCGTGATAGCTTAAAAAAGGACCT GAATGCGACCGCCTTGGGCTTTCGGGCTTTACGCTTACACCGTTATAAT GTAAGCTCAGGAGTGCTGGAGAACTTCCGTGATGACAATGGTCAATTC TTTTGCGGTTCTACTGTGGAGGAGGAAGGCGCGGAGGCCTACAATAAA CATGTACGTTGCATGCTGTCCCTGTCCCGCGCTTCCAATATTTTATTCC CGGGCGAGAAAGTGATGGAAGAAGCGAAGGCGTTTACGACCAACTAT CTTAAGAAAGTCCTGGCGGGTCGTGAAGCAACTCATGTCGACGAGAGT CTCCTTGGAGAGGTCAAGTATGCACTAGAATTTCCGTGGCATTGTTCC GTGCAGCGCTGGGAGGCACGTTCTTTTATCGAAATTTTCGGTCAGATT GATAGTGAACTGAAAAGCAACCTCTCTAAAAAAATGCTCGAACTCGC AAAACTTGATTTTAACATACTCCAGTGTACGCATCAAAAAGAGCTCCA GATCATTAGTCGATGGTTCGCCGATTCAAGTATCGCAAGTCTGAACTT TTACCGTAAATGCTATGTGGAATTTTACTTCTGGATGGCCGCGGCAATT TCAGAACCAGAATTTAGTGGCTCTCGCGTGGCATTCACTAAAATTGCG ATCTTGATGACAATGTTAGATGACTTATACGACACGCATGGGACGCTG GATCAATTGAAAATATTTACCGAAGGTGTGCGCAGGTGGGACGTGTCG CTGGTGGAGGGCCTGCCGGATTTCATGAAAATTGCCTTTGAGTTCTGG TTAAAGACCTCCAACGAACTGATTGCGGAGGCGGTTAAGGCCCAAGG CCAGGATATGGCGGCCTATATCCGCAAAAACGCTTGGGAACGCTATCT GGAAGCGTATTTGCAGGATGCCGAATGGATCGCCACCGGTCACGTTCC GACATTCGATGAATATCTGAACAATGGCACCCCCAACACCGGTATGTG TGTACTTAATCTGATCCCGTTGCTGCTTATGGGCGAACACTTGCCGATC GATATTCTTGAACAGATCTTTCTGCCGAGCCGGTTCCACCATCTGATTG AACTGGCTAGCCGACTGGTCGATGATGCGAGAGATTTTCAAGCCGAAA AAGATCATGGTGATTTATCCTGCATCGAATGCTACCTGAAAGACCATC CGGAATCAACAGTTGAAGACGCCCTGAATCACGTCAACGGCCTGCTGG GGAATTGTTTGCTGGAAATGAATTGGAAATTTCTGAAAAAACAGGACT CGGTACCTCTGTCGTGTAAAAAATACTCATTCCACGTCCTGGCGCGGT CGATTCAGTTTATGTATAACCAGGGGGACGGGTTTTCGATTTCGAACA AAGTTATTAAAGACCAGGTCCAGAAAGTTCTAATCGTTCCGGTTCCTA TATAA ABS Abies TGAAACGAGAATTTCCTCCAGGATTTTGGAAGGATGATCTTATCGATT SEQ grandis CTCTAACGTCATCTCACAAGGTTGCAGCATCAGACGAGAAGCGTATCG ID AGACATTAATATCCGAGATTAAGAATATGTTTAGATGTATGGGCTATG No.89 GCGAAACGAATCCCTCTGCATATGACACTGCTTGGGTAGCAAGGATTC CAGCAGTTGATGGCTCTGACAACCCTCACTTTCCTGAGACGGTTGAAT GGATTCTTCAAAATCAGTTGAAAGATGGGTCTTGGGGTGAAGGATTCT ACTTCTTGGCATATGACAGAATACTGGCTACACTTGCATGTATTATTAC CCTTACCCTCTGGCGTACTGGGGAGACACAAGTACAGAAAGGTATTGA ATTCTTCAGGACACAAGCTGGAAAGATGGAAGATGAAGCTGATAGTC ATAGGCCAAGTGGATTTGAAATAGTATTTCCTGCAATGCTAAAGGAAG CTAAAATCTTAGGCTTGGATCTGCCTTACGATTTGCCATTCCTGAAACA AATCATCGAAAAGCGGGAGGCTAAGCTTAAAAGGATTCCC ACTGATGTTCTCTATGCCCTTCCAACAACGTTATTGTATTCTTTGGAAG GTTTACAAGAAATAGTAGACTGGCAGAAAATAATGAAACTTCAATCC AAGGATGGATCATTTCTCAGCTCTCCGGCATCTACAGCGGCTGTATTC ATGCGTACAGGGAACAAAAAGTGCTTGGATTTCTTGAACTTTGTCTTG AAGAAATTCGGAAACCATGTGCCTTGTCACTATCCGCTTGATCTATTTG AACGTTTGTGGGCGGTTGATACAGTTGAGCGGCTAGGTATCGATCGTC ATTTCAAAGAGGAGATCAAGGAAGCATTGGATTATGTTTACAGCCATT GGGACGAAAGAGGCATTGGATGGGCGAGAGAGAATCCTGTTCCTGAT ATTGATGATACAGCCATGGGCCTTCGAATCTTGAGATTACATGGATAC AATGTATCCTCAGATGTTTTAAAAACATTTAGAGATGAGAATGGGGAG TTCTTTTGCTTCTTGGGTCAAACACAGAGAGGAGTTACAGACATGTTA AACGTCAATCGTTGTTCACATGTTTCATTTCCGGGAGAAACGATCATG GAAGAAGCAAAACTCTGTACCGAAAGGTATCTGAGGAATGCTCTGGA AAATGTGGATGCCTTTGACAAATGGGCTTTTAAAAAGAATATTCGGGG AGAGGTAGAGTATGCACTCAAATATCCCTGGCATAAGAGTATGCCAA GGTTGGAGGCTAGAAGCTATATTGAAAACTATGGGCCAGATGATGTGT GGCTTGGAAAAACTGTATATATGATGCCATACATTTCGAATGAAAAGT ATTTAGAACTAGCGAAACTGGACTTCAATAAGGTGCAGTCTATACACC AAACAGAGCTTCAAGATCTTCGAAGGTGGTGGAAATCATCCGGTTTCA CGGATCTGAATTTCACTCGTGAGCGTGTGACGGAAATATATTTCTCAC CGGCATCCTTTATCTTTGAGCCCGAGTTTTCTAAGTGCAGAGAGGTTTA TACAAAAACTTCCAATTTCACTGTTATTTTAGATGATCTTTATGACGCC CATGGATCTTTAGACGATCTTAAGTTGTTCACAGAATCAGTCAAAAGA TGGGATCTATCACTAGTGGACCAAATGCCACAACAAATGAAAATATGT TTTGTGGGTTTCTACAATACTTTTAATGATATAGCAAAAGAAGGACGT GAGAGGCAAGGGCGCGATGTGCTAGGCTACATTCAAAATGTTTGGAA AGTCCAACTTGAAGCTTACACGAAAGAAGCAGAATGGTCTGAAGCTA AATATGTGCCATCCTTCAATGAATACATAGAGAATGCGAGTGTGTCAA TAGCATTGGGAACAGTCGTTCTCATTAGTGCTCTTTTCACTGGGGAGGT TCTTACAGATGAAGTACTCTCCAAAATTGATCGCGAATCTAGATTTCTT CAACTCATGGGCTTAACAGGGCGTTTGGTGAATGACACCAAAACTTAT CAGGCAGAGAGAGGTCAAGGTGAGGTGGCTTCTGCCATACAATGTTAT ATGAAGGACCATCCTAAAATCTCTGAAGAAGAAGCTCTACAACATGTC TATAGTGTCATGGAAAATGCCCTCGAAGAGTTGAATAGGGAGTTTGTG AATAACAAAATACCGGATATTTACAAAAGACTGGTTTTTGAAACTGCA AGAATAATGCAACTCTTTTATATGCAAGGGGATGGTTTGACACTATCA CATGATATGGAAATTAAAGAGCATGTCAAAAATTGCCTCTTCCAACCA GTTGCC TXS Taxus ATGAGCAGCAGCACTGGCACTAGCAAGGTGGTTTCCGAGACTTCCAGT SEQ brevifola ACCATTGTGGATGATATCCCTCGACTCTCCGCCAATTATCATGGCGATC ID TGTGGCACCACAATGTTATACAAACTCTGGAGACACCGTTTCGTGAGA No.90 GTTCTACTTACCAAGAACGGGCAGATGAGCTGGTTGTGAAAATTAAAG ATATGTTCAATGCGCTCGGAGACGGAGATATCAGTCCGTCTGCATACG ACACTGCGTGGGTGGCGAGGCTGGCGACCATTTCCTCTGATGGATCTG AGAAGCCACGGTTTCCTCAGGCCCTCAACTGGGTTTTCAACAACCAGC TCCAGGATGGATCGTGGGGTATCGAATCGCACTTTAGTTTATGCGATC GATTGCTTAACACGACCAATTCTGTTATCGCCCTCTCGGTTTGGAAAAC AGGGCACAGCCAAGTACAACAAGGTGCTGAGTTTATTGCAGAGAATC TAAGATTACTCAATGAGGAAGATGAGTTGTCCCCGGATTTCCAAATAA TCTTTCCTGCTCTGCTGCAAAAGGCAAAAGCGTTGGGGATCAATCTTC CTTACGATCTTCCATTTATCAAATATTTGTCGACAACACGGGAAGCCA GGCTTACAGATGTTTCTGCGGCAGCAGACAATATTCCAGCCAACATGT TGAATGCGTTGGAAGGACTCGAGGAAGTTATTGACTGGAACAAGATT ATGAGGTTTCAAAGTAAAGATGGATCTTTCCTGAGCTCCCCTGCCTCC ACTGCCTGTGTACTGATGAATACAGGGGACGAAAAATGTTTCACTTTT CTCAACAATCTGCTCGACAAATTCGGCGGCTGCGTGCCCTGTATGTAT TCCATCGATCTGCTGGAACGCCTTTCGCTGGTTGATAACATTGAGCATC TCGGAATCGGTCGCCATTTCAAACAAGAAATCAAAGGAGCTCTTGATT ATGTCTACAGACATTGGAGTGAAAGGGGCATCGGTTGGGGCAGAGAC AGCCTTGTTCCAGATCTCAACACCACAGCCCTCGGCCTGCGAACTCTT CGCATGCACGGATACAATGTTTCTTCAGACGTTTTGAATAATTTCAAA GATGAAAACGGGCGGTTCTTCTCCTCTGCGGGCCAAACCCATGTCGAA TTGAGAAGCGTGGTGAATCTTTTCAGAGCTTCCGACCTTGCATTTCCTG ACGAAAGAGCTATGGACGATGCTAGAAAATTTGCAGAACCATATCTTA GAGAGGCACTTGCAACGAAAATCTCAACCAATACAAAACTATTCAAA GAGATTGAGTACGTGGTGGAGTACCCTTGGCACATGAGTATCCCACGC TTAGAAGCCAGAAGTTATATTGATTCATATGACGACAATTATGTATGG CAGAGGAAGACTCTATATAGAATGCCATCTTTGAGTAATTCAAAATGT TTAGAATTGGCAAAATTGGACTTCAATATCGTACAATCTTTGCATCAA GAGGAGTTGAAGCTTCTAACAAGATGGTGGAAGGAATCCGGCATGGC AGATATAAATTTCACTCGACACCGAGTGGCGGAGGTTTATTTTTCATC AGCTACATTTGAACCCGAATATTCTGCCACTAGAATTGCCTTCACAAA AATTGGTTGTTTACAAGTCCTTTTTGATGATATGGCTGACATCTTTGCA ACACTAGATGAATTGAAAAGTTTCACTGAGGGAGTAAAGAGATGGGA TACATCTTTGCTACATGAGATTCCAGAGTGTATGCAAACTTGCTTTAAA GTTTGGTTCAAATTAATGGAAGAAGTAAATAATGATGTGGTTAAGGTA CAAGGACGTGACATGCTCGCTCACATAAGAAAACCCTGGGAGTTGTAC TTCAATTGTTATGTACAAGAAAGGGAGTGGCTTGAAGCCGGGTATATA CCAACTTTTGAAGAGTACTTAAAGACTTATGCTATATCAGTAGGCCTT GGACCGTGTACCCTACAACCAATACTACTAATGGGTGAGCTTGTGAAA GATGATGTTGTTGAGAAAGTGCACTATCCCTCAAATATGTTTGAGCTT GTATCCTTGAGCTGGCGACTAACAAACGACACCAAAACATATCAGGCT GAAAAGGCTCGAGGACAACAAGCCTCAGGCATAGCATGCTATATGAA GGATAATCCAGGAGCAACTGAGGAAGATGCCATTAAGCACATATGTC GTGTTGTTGATCGGGCCTTGAAAGAAGCAAGCTTTGAATATTTCAAAC CATCCAATGATATCCCAATGGGTTGCAAGTCCTTTATTTTTAACCTTAG ATTGTGTGTCCAAATCTTTTACAAGTTTATAGATGGGTACGGAATCGC CAATGAGGAGATTAA GGACTATATAAGAAAAGTTTATATTGATCCAATTCAAGTATGA GGPPS Taxus ATGTTTGATTTCAATGAATATATGAAAAGTAAGGCTGTTGCGGTAGAC SEQ Canadensis GCGGCTCTGGATAAAGCGATTCCGCTGGAATATCCCGAGAAGATTCAC ID GAATCGATGCGCTACTCCCTGTTAGCAGGAGGGAAACGCGTTCGTCCG No.91 GCATTATGCATCGCGGCCTGTGAACTCGTCGGCGGTTCACAGGACTTA GCAATGCCAACTGCTTGCGCAATGGAAATGATTCACACAATGAGCCTG ATTCATGATGATTTGCCTTGCATGGACAACGATGACTTTCGGCGCGGT AAACCTACTAATCATAAGGTTTTTGGCGAAGATACTGCAGTGCTGGCG GGCGATGCGCTGCTGTCGTTTGCCTTCGAACATATCGCCGTCGCGACC TCGAAAACCGTCCCGTCGGACCGTACGCTTCGCGTGATTTCCGAGCTG GGAAAGACCATCGGCTCTCAAGGACTCGTGGGTGGTCAGGTAGTTGAT ATCACGTCTGAGGGTGACGCGAACGTGGACCTGAAAACCCTGGAGTG GATCCATATTCACAAAACGGCCGTGCTGCTGGAATGTAGCGTGGTGTC AGGGGGGATCTTGGGGGGCGCCACGGAGGATGAAATCGCGCGTATTC GTCGTTATGCCCGCTGTGTTGGACTGTTATTTCAGGTGGTGGATGACAT CCTGGATGTCACAAAATCCAGCGAAGAGCTTGGCAAGACCGCGGGCA AAGACCTTCTGACGGATAAGGCTACATACCCGAAATTGATGGGCTTGG AGAAAGCCAAGGAGTTCGCAGCTGAACTTGCCACGCGGGCGAAGGAA GAACTCTCTTCTTTCGATCAAATCAAAGCCGCGCCACTGCTGGGCCTC GCCGATTACATTGCGTTTCGTCAGAACTGA P450.sub.BM3 Bacillus ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAA SEQ megaterium AAATTTACCGTTATTAAACACAGATAAACCGGTTCAAGCTTTGATGAA ID AATTGCGGATGAATTAGGAGAAATCTTTAAATTCGAGGCGCCTGGTCG No.92 TGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAGCATGCGA TGAATCACGCTTTGATAAAAACTTAAGTCAAGCGCTTAAATTTGTACG TGATTTTGCAGGAGACGGGTTATTTACAAGCTGGACGCATGAAAAAAA TTGGAAAAAAGCGCATAATATCTTACTTCCAAGCTTCAGTCAGCAGGC AATGAAAGGCTATCATGCGATGATGGTCGATATCGCCGTGCAGCTTGT TCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATTGAAGTACCGG AAGACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTA ACTATCGCTTTAACAGCTTTTACCGAGATCAGCCTCATCCATTTATTAC AAGTATGGTCCGTGCACTGGATGAAGCAATGAACAAGCTGCAGCGAG CAAATCCAGACGACCCAGCTTATGATGAAAACAAGCGCCAGTTTCAA GAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATTATTGCAGA TCGCAAAGCAAGCGGTGAACAAAGCGATGATTTATTAACGCATATGCT AAACGGAAAAGATCCAGAAACGGGTGAGCCGCTTGATGACGAGAACA TTCGCTATCAAATTATTACATTCTTAATTGCGGGACACGAAACAACAA GTGGTCTTTTATCATTTGCGCTGTATTTCTTAGTGAAAAATCCACATGT ATTACAAAAAGCAGCAGAAGAAGCAGCACGAGTTCTAGTAGATCCTG TTCCAAGCTACAAACAAGTCAAACAGCTTAAATATGTCGGCATGGTCT TAAACGAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGTTTTCCCTATA TGCAAAAGAAGATACGGTGCTTGGAGGAGAATATCCTTTAGAAAAAG GCGACGAACTAATGGTTCTGATTCCTCAGCTTCACCGTGATAAAACAA TTTGGGGAGACGATGTGGAAGAGTTCCGTCCAGAGCGTTTTGAAAATC CAAGTGCGATTCCGCAGCATGCGTTTAAACCGTTTGGAAACGGTCAGC GTGCGTGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGTAC TTGGTATGATGCTAAAACACTTTGACTTTGAAGATCATACAAACTACG AGCTGGATATTAAAGAAACTTTAACGTTAAAACCTGAAGGCTTTGTGG TAAAAGCAAAATCGAAAAAAATTCCGCTTGGCGGTATTCCTTCACCTA GCACTGAACAGTCTGCTAAAAAAGTACGCAAAAAGGCAGAAAACGCT CATAATACGCCGCTGCTTGTGCTATACGGTTCAAATATGGGAACAGCT GAAGGAACGGCGCGTGATTTAGCAGATATTGCAATGAGCAAAGGATT TGCACCGCAGGTCGCAACGCTTGATTCACACGCCGGAAATCTTCCGCG CGAAGGAGCTGTATTAATTGTAACGGCGTCTTATAACGGTCATCCGCC TGATAACGCAAAGCAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGA TGAAGTAAAAGGCGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAA CTGGGCTACTACGTATCAAAAAGTGCCTGCTTTTATCGATGAAACGCT TGCCGCTAAAGGGGCAGAAAACATCGCTGACCGCGGTGAAGCAGATG CAAGCGACGACTTTGAAGGCACATATGAAGAATGGCGTGAACATATG TGGAGTGACGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAA GATAATAAATCTACTCTTTCACTTCAATTTGTCGACAGCGCCGCGGAT ATGCCGCTTGCGAAAATGCACGGTGCGTTTTCAACGAACGTCGTAGCA AGCAAAGAACTTCAACAGCCAGGCAGTGCACGAAGCACGCGACATCT TGAAATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCATTT AGGTGTTATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGC AAGGTTCGGCCTAGATGCATCACAGCAAATCCGTCTGGAAGCAGAAG AAGAAAAATTAGCTCATTTGCCACTCGCTAAAACAGTATCCGTAGAAG AGCTTCTGCAATACGTGGAGCTTCAAGATCCTGTTACGCGCACGCAGC TTCGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGAGC TTGAAGCCTTGCTTGAAAAGCAAGCCTACAAAGAACAAGTGCTGGCA AAACGTTTAACAATGCTTGAACTGCTTGAAAAATACCCGGCGTGTGAA ATGAAATTCAGCGAATTTATCGCCCTTCTGCCAAGCATACGCCCGCGC TATTACTCGATTTCTTCATCACCTCGTGTCGATGAAAAACAAGCAAGC ATCACGGTCAGCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAGA ATATAAAGGAATTGCGTCGAACTATCTTGCCGAGCTGCAAGAAGGAG ATACGATTACGTGCTTTATTTCCACACCGCAGTCAGAATTTACGCTGCC AAAAGACCCTGAAACGCCGCTTATCATGGTCGGACCGGGAACAGGCG TCGCGCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAGCTAAAAGAAC AAGGACAGTCACTTGGAGAAGCACATTTATACTTCGGCTGCCGTTCAC CTCATGAAGACTATCTGTATCAAGAAGAGCTTGAAAACGCCCAAAGC GAAGGCATCATTACGCTTCATACCGCTTTTTCTCGCATGCCAAATCAGC CGAAAACATACGTTCAGCACGTAATGGAACAAGACGGCAAGAAATTG ATTGAACTTCTTGATCAAGGAGCGCACTTCTATATTTGCGGAGACGGA AGCCAAATGGCACCTGCCGTTGAAGCAACGCTTATGAAAAGCTATGCT GACGTTCACCAAGTGAGTGAAGCAGACGCTCGCTTATGGCTGCAGCAG CTAGAAGAAAAAGGCCGATACGCAAAAGACGTGTGGGCTGGGTAA SpecR Bacterial ATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGT SEQ AGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACA ID TTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATAT No.93 TGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCG AGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAG CGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACAT CATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATG GCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGA CATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGC CTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACA GGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCC GCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCG CATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCG CTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCA TACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGG CCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCG AGATCACCAAGGTAGTCGGCAAA LOV2 Avena TTGGCTACTACACTTGAACGTATTGAGAAGAACTTTGTCATTACTGAC SEQ sativa CCAAGGTTGCCAGATAATCCCATTATATTCGCGTCCGATAGTTTCTTGC ID AGTTGACAGAATATAGCCGTGAAGAAATTTTGGGAAGAAACTGCAGG No.94 TTTCTACAAGGTCCTGAAACTGATCGCGCGACAGTGAGAAAAATTAGA GATGCCATAGATAACCAAACAGAGGTCACTGTTCAGCTGATTAATTAT ACAAAGAGTGGTAAAAAGTTCTGGAACCTCTTTCACTTGCAGCCTATG CGAGATCAGAAGGGAGATGTCCAGTACTTTATTGGGGTTCAGTTGGAT GGAACTGAGCATGTCCGAGATGCTGCCGAGAGAGAGGGAGTCATGCT GATTAAGAAAACTGCAGAAAATATTGATGAGGCGGCAAAAGAACTTC CA BphP1 Rhodo- ATGGCTAGCGTGGCAGGTCATGCCTCTGGCAGCCCCGCATTCGGGACC SEQ pseudomonas GCCGATCTTTCGAATTGCGAACGTGAAGAGATCCACCTCGCCGGCTCG ID palustris ATCCAGCCGCATGGCGCGCTTCTGGTCGTCAGCGAGCCGGATCATCGC No.95 ATCATCCAGGCCAGCGCCAACGCCGCGGAATTTCTGAATCTCGGAAGC GTGCTCGGCGTTCCGCTCGCCGAGATCGACGGCGATCTGTTGATCAAG ATCCTGCCGCATCTCGATCCCACCGCCGAAGGCATGCCGGTCGCGGTG CGCTGCCGGATCGGCAATCCCTCCACGGAGTACGACGGTCTGATGCAT CGGCCTCCGGAAGGCGGGCTGATCATCGAGCTCGAACGTGCCGGCCC GCCGATCGATCTGTCCGGCACGCTGGCGCCGGCGCTGGAGCGGATCCG CACGGCGGGCTCGCTGCGCGCGCTGTGCGATGACACCGCGCTGCTGTT TCAGCAGTGCACCGGCTACGACCGGGTGATGGTGTATCGCTTCGACGA GCAGGGCCACGGCGAAGTGTTCTCCGAGCGCCACGTGCCCGGGCTCG AATCCTATTTCGGCAACCGCTATCCGTCGTCGGACATTCCGCAGATGG CGCGGCGGCTGTACGAGCGGCAGCGCGTCCGCGTGCTGGTCGACGTCA GCTATCAGCCGGTGCCGCTGGAGCCGCGGCTGTCGCCGCTGACCGGGC GCGATCTCGACATGTCGGGCTGCTTCCTGCGCTCGATGTCGCCGATCC ATCTGCAGTACCTGAAGAACATGGGCGTGCGCGCCACCCTGGTGGTGT CGCTGGTGGTCGGCGGCAAGCTGTGGGGCCTGGTTGCCTGTCATCATT ATCTGCCGCGCTTCATGCATTTCGAGCTGCGGGCGATCTGCGAACTGC TCGCCGAAGCGATCGCGACGCGGATCACCGCGCTTGAGAGCTTCGCGC AGAGCCAGTCGGAGCTGTTCGTGCAGCGGCTCGAACAGCGCATGATC GAAGCGATTACCCGTGAAGGCGATTGGCGCGCAGCGATTTTCGACACC AGCCAATCGATCCTGCAGCCGCTGCACGCCGCCGGTTGCGCGCTGGTG TACGAAGACCAGATCAGGACCATCGGCGACGTGCCTTCCACGCAGGA TGTGCGCGAGATCGCCGGGTGGCTCGATCGCCAGCCGCGCGCGGCGGT GACCTCGACCGCGTCGCTCGGTCTCGACGTGCCGGAGCTCGCGCATCT GACGCGGATGGCGAGCGGCGTGGTCGCGGCGCCGATTTCGGATCATC GCGGCGAGTTTCTGATGTGGTTCCGCCCCGAGCGCGTCCACACCGTTA CCTGGGGCGGCGATCCGAAGAAGCCGTTCACGATGGGCGATACACCG GCGGATCTGTCGCCGCGGCGCTCCTTCGCCAAATGGCATCAGGTTGTC GAAGGCACGTCCGATCCGTGGACGGCCGCCGATCTCGCCGCGGCTCGC ACCATCGGTCAGACCGTCGCCGACATCGTGCTGCAATTCCGCGCGGTG CGGACACTGATCGCCCGCGAACAGTACGAACAGTTTTCGTCCCAGGTG CACGCTTCGATGCAGCCGGTGCTGATCACCGACGCCGAAGGCCGCATC CTGCTGATGAACGACTCGTTCCGCGACATGTTGCCGGCGGGTTCGCCA TCCGCCGTCCATCTCGACGATCTCGCCGGGTTCTTCGTCGAATCGAAC GATTTCCTGCGCAACGTCGCCGAACTGATCGATCACGGCCGCGGGTGG CGCGGCGAAGTTCTGCTGCGCGGCGCAGGCAACCGCCCGTTGCCGCTG GCAGTGCGCGCCGATCCGGTGACGCGCACGGAGGACCAGTCGCTCGG CTTCGTGCTGATCTTCAGCGACGCTACCGATCGTCGCACCGCAGATGC CGCACGCACGCGTTTCCAGGAAGGCATTCTTGCCAGCGCACGTCCCGG CGTGCGGCTCGACTCCAAGTCCGACCTGTTGCACGAGAAGCTGCTGTC CGCGCTGGTCGAGAACGCGCAGCTTGCCGCATTGGAAATCACTTACGG CGTCGAGACCGGACGCATCGCCGAGCTGCTCGAAGGCGTCCGCCAGTC GATGCTGCGCACCGCCGAAGTGCTCGGCCATCTGGTGCAGCACGCGGC GCGCACGGCCGGCAGCGACAGCTCGAGCAATGGCTCGCAGAACAAGA AGGAATTCGATAGTGCTGGTAGTGCTGGTAGTGCTGGTACTAGT PTP1B.sub.1-435 H. ATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAGTCCGGGAGCTG SEQ Sapiens GGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATG ID TAGAGTGGCCAAGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAG No.96 ACGTCAGTCCCTTTGACCATAGTCGGATTAAACTACATCAAGAAGATA ATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGG AGTTACATTCTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTT GGGAGATGGTGTGGGAGCAGAAAAGCAGGGGTGTCGTCATGCTCAAC AGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACA AAAAGAAGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAA CATTGATCTCTGAAGATATCAAGTCATATTATACAGTGCGACAGCTAG AATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCC ACTATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCAT TCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGCCCGG AGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTG GAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGA AAGACCCTTCTTCCGTTGATATCAAGAAAGTGCTGTTAGAAATGAGGA AGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTTCTCCT ACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCG TGCAGGATCAGTGGAAGGAGCTTTCCCACGAGGACCTGGAGCCCCCA CCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAATCCTGGAG CCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTG AAGGAAGAGACCCAGGAGGATAAAGACTGCCCCATCAAGGAAGAAA AAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAAAGCATGAGT CAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGG TGCCCAGGCTGCCTCCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGA AGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTTCCTGGTCAACA TGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGT TCCTGTTCAACAGCAACACATAG TC- H. ATGCCCACCACCATCGAGCGGGAGTTCGAAGAGTTGGATACTCAGCGT SEQ PTP Sapiens CGCTGGCAGCCGCTGTACTTGGAAATTCGAAATGAGTCCCATGACTAT ID (full) CCTCATAGAGTGGCCAAGTTTCCAGAAAACAGAAATCGAAACAGATA No.97 CAGAGATGTAAGCCCATATGATCACAGTCGTGTTAAACTGCAAAATGC TGAGAATGATTATATTAATGCCAGTTTAGTTGACATAGAAGAGGCACA AAGGAGTTACATCTTAACACAGGGTCCACTTCCTAACACATGCTGCCA TTTCTGGCTTATGGTTTGGCAGCAGAAGACCAAAGCAGTTGTCATGCT GAACCGCATTGTGGAGAAAGAATCGGTTAAATGTGCACAGTACTGGC CAACAGATGACCAAGAGATGCTGTTTAAAGAAACAGGATTCAGTGTG AAGCTCTTGTCAGAAGATGTGAAGTCGTATTATACAGTACATCTACTA CAATTAGAAAATATCAATAGTGGTGAAACCAGAACAATATCTCACTTT CATTATACTACCTGGCCAGATTTTGGAGTCCCTGAATCACCAGCTTCAT TTCTCAATTTCTTGTTTAAAGTGAGAGAATCTGGCTCCTTGAACCCTGA CCATGGGCCTGCGGTGATCCACTGTAGTGCAGGCATTGGGCGCTCTGG CACCTTCTCTCTGGTAGACACTTGTCTTGTTTTGATGGAAAAAGGAGAT GATATTAACATAAAACAAGTGTTACTGAACATGAGAAAATACCGAAT GGGTCTTATTCAGACCCCAGATCAACTGAGATTCTCATACATGGCTAT AATAGAAGGAGCAAAATGTATAAAGGGAGATTCTAGTATACAGAAAC GATGGAAAGAACTTTCTAAGGAAGACTTATCTCCTGCCTTTGATCATT CACCAAACAAAATAATGACTGAAAAATACAATGGGAACAGA PTPN5 H.sapiens ATGTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCC SEQ ATGTCCCGTGTCCTCCAAGCAGAAGAGCTTCATGAAAAGGCCCTGGAC ID CCTTTCCTGCTGCAGGCGGAATTCTTTGAAATCCCCATGAACTTTGTGG No.98 ATCCGAAAGAGTACGACATCCCTGGGCTGGTGCGGAAGAACCGGTAC AAAACCATACTTCCCAACCCTCACAGCAGAGTGTGTCTGACCTCACCA GACCCTGACGACCCTCTGAGTTCCTACATCAATGCCAACTACATCCGG GGCTATGGTGGGGAGGAGAAGGTGTACATCGCCACTCAGGGACCCAT CGTCAGCACGGTCGCCGACTTCTGGCGCATGGTGTGGCAGGAGCACAC GCCCATCATTGTCATGATCACCAACATCGAGGAGATGAACGAGAAAT GCACCGAGTATTGGCCGGAGGAGCAGGTGGCGTACGACGGTGTTGAG ATCACTGTGCAGAAAGTCATTCACACGGAGGATTACCGGCTGCGACTC ATCTCCCTCAAGAGTGGGACTGAGGAGCGAGGCCTGAAGCATTACTG GTTCACATCCTGGCCCGACCAGAAGACCCCAGACCGGGCCCCCCCACT CCTGCACCTGGTGCGGGAGGTGGAGGAGGCAGCCCAGCAGGAGGGGC CCCACTGTGCCCCCATCATCGTCCACTGCAGTGCAGGGATTGGGAGGA CCGGCTGCTTCATTGCCACCAGCATCTGCTGCCAGCAGCTGCGGCAGG AGGGTGTAGTGGACATCCTGAAGACCACGTGCCAGCTCCGTCAGGAC AGGGGCGGCATGATCCAGACATGCGAGCAGTACCAGTTTGTGCACCA CGTCATGAGCCTCTACGAAAAGCAGCTGTCCCACCAGTCCTGA PTPN6 H.sapiens ATGGTGAGGTGGTTTCACCGAGACCTCAGTGGGCTGGATGCAGAGACC SEQ CTGCTCAAGGGCCGAGGTGTCCACGGTAGCTTCCTGGCTCGGCCCAGT ID CGCAAGAACCAGGGTGACTTCTCGCTCTCCGTCAGGGTGGGGGATCAG No.99 GTGACCCATATTCGGATCCAGAACTCAGGGGATTTCTATGACCTGTAT GGAGGGGAGAAGTTTGCGACTCTGACAGAGCTGGTGGAGTACTACAC TCAGCAGCAGGGTGTGGTGCAGGACCGCGACGGCACCATCATCCACCT CAAGTACCCGCTGAACTGCTCCGATCCCACTAGTGAGAGGTGGTACCA TGGCCACATGTCTGGCGGGCAGGCAGAGACGCTGCTGCAGGCCAAGG GCGAGCCCTGGACGTTTCTTGTGCGTGAGAGCCTCAGCCAGCCTGGAG ACTTCGTGCTTTCTGTGCTCAGTGACCAGCCCAAGGCTGGCCCAGGCT CCCCGCTCAGGGTCACCCACATCAAGGTCATGTGCGAGGGTGGACGCT ACACAGTGGGTGGTTTGGAGACCTTCGACAGCCTCACGGACCTGGTGG AGCATTTCAAGAAGACGGGGATTGAGGAGGCCTCAGGCGCCTTTGTCT ACCTGCGGCAGCCGTACTATGCCACGAGGGTGAATGCGGCTGACATTG AGAACCGAGTGTTGGAACTGAACAAGAAGCAGGAGTCCGAGGATACA GCCAAGGCTGGCTTCTGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGA GGTGAAGAACTTGCACCAGCGTCTGGAAGGGCAACGGCCAGAGAACA AGGGCAAGAACCGCTACAAGAACATTCTCCCCTTTGACCACAGCCGAG TGATCCTGCAGGGACGGGACAGTAACATCCCCGGGTCCGACTACATCA ATGCCAACTACATCAAGAACCAGCTGCTAGGCCCTGATGAGAACGCTA AGACCTACATCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAATGACT TCTGGCAGATGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATGACCA CCCGAGAGGTGGAGAAAGGCCGGAACAAATGCGTCCCATACTGGCCC GAGGTGGGCATGCAGCGTGCTTATGGGCCCTACTCTGTGACCAACTGC GGGGAGCATGACACAACCGAATACAAACTCCGTACCTTACAGGTCTCC CCGCTGGACAATGGAGACCTGATTCGGGAGATCTGGCATTACCAGTAC CTGAGCTGGCCCGACCATGGGGTCCCCAGTGAGCCTGGGGGTGTCCTC AGCTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTCTGCCTCACGCA GGGCCCATCATCGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCACC ATCATTGTCATCGACATGCTCATGGAGAACATCTCCACCAAGGGCCTG GACTGTGACATTGACATCCAGAAGACCATCCAGATGGTGCGGGCGCA GCGCTCGGGCATGGTGCAGACGGAGGCGCAGTACAAGTTCATCTACGT GGCCATCGCCCAGTTCATTGAAACCACTAAGAAGAAGCTGGAGGTCCT GCAGTCGCAGAAGGGCCAGGAGTCGGAGTACGGGAACATCACCTATC CCCCAGCCATGAAGAATGCCCATGCCAAGGCCTCCCGCACCTCGTCCA AACACAAGGAGGATGTGTATGAGAACCTGCACACTAAGAACAAGAGG GAGGAGAAAGTGAAGAAGCAGCGGTCAGCAGACAAGGAGAAGAGCA AGGGTTCCCTCAAGAGGAAGTGA PTPN11 H.sapiens ATGACATCGCGGAGATGGTTTCACCCAAATATCACTGGTGTGGAGGCA SEQ GAAAACCTACTGTTGACAAGAGGAGTTGATGGCAGTTTTTTGGCAAGG ID CCTAGTAAAAGTAACCCTGGAGACTTCACACTTTCCGTTAGAAGAAAT No. GGAGCTGTCACCCACATCAAGATTCAGAACACTGGTGATTACTATGAC 100 CTGTATGGAGGGGAGAAATTTGCCACTTTGGCTGAGTTGGTCCAGTAT TACATGGAACATCACGGGCAATTAAAAGAGAAGAATGGAGATGTCAT TGAGCTTAAATATCCTCTGAACTGTGCAGATCCTACCTCTGAAAGGTG GTTTCATGGACATCTCTCTGGGAAAGAAGCAGAGAAATTATTAACTGA AAAAGGAAAACATGGTAGTTTTCTTGTACGAGAGAGCCAGAGCCACC CTGGAGATTTTGTTCTTTCTGTGCGCACTGGTGATGACAAAGGGGAGA GCAATGACGGCAAGTCTAAAGTGACCCATGTTATGATTCGCTGTCAGG AACTGAAATACGACGTTGGTGGAGGAGAACGGTTTGATTCTTTGACAG ATCTTGTGGAACATTATAAGAAGAATCCTATGGTGGAAACATTGGGTA CAGTACTACAACTCAAGCAGCCCCTTAACACGACTCGTATAAATGCTG CTGAAATAGAAAGCAGAGTTCGAGAACTAAGCAAATTAGCTGAGACC ACAGATAAAGTCAAACAAGGCTTTTGGGAAGAATTTGAGACACTACA ACAACAGGAGTGCAAACTTCTCTACAGCCGAAAAGAGGGTCAAAGGC AAGAAAACAAAAACAAAAATAGATATAAAAACATCCTGCCCTTTGAT CATACCAGGGTTGTCCTACACGATGGTGATCCCAATGAGCCTGTTTCA GATTACATCAATGCAAATATCATCATGCCTGAATTTGAAACCAAGTGC AACAATTCAAAGCCCAAAAAGAGTTACATTGCCACACAAGGCTGCCT GCAAAACACGGTGAATGACTTTTGGCGGATGGTGTTCCAAGAAAACTC CCGAGTGATTGTCATGACAACGAAAGAAGTGGAGAGAGGAAAGAGTA AATGTGTCAAATACTGGCCTGATGAGTATGCTCTAAAAGAATATGGCG TCATGCGTGTTAGGAACGTCAAAGAAAGCGCCGCTCATGACTATACGC TAAGAGAACTTAAACTTTCAAAGGTTGGACAAGGGAATACGGAGAGA ACGGTCTGGCAATACCACTTTCGGACCTGGCCGGACCACGGCGTGCCC AGCGACCCTGGGGGCGTGCTGGACTTCCTGGAGGAGGTGCACCATAA GCAGGAGAGCATCATGGATGCAGGGCCGGTCGTGGTGCACTGCAGTG CTGGAATTGGCCGGACAGGGACGTTCATTGTGATTGATATTCTTATTG ACATCATCAGAGAGAAAGGTGTTGACTGCGATATTGACGTTCCCAAAA CCATCCAGATGGTGCGGTCTCAGAGGTCAGGGATGGTCCAGACAGAA GCACAGTACCGATTTATCTATATGGCGGTCCAGCATTATATTGAAACA CTACAGCGCAGGATTGAAGAAGAGCAGAAAAGCAAGAGGAAAGGGC ACGAATATACAAATATTAAGTATTCTCTAGCGGACCAGACGAGTGGAG ATCAGAGCCCTCTCCCGCCTTGTACTCCAACGCCACCCTGTGCAGAAA TGAGAGAAGACAGTGCTAGAGTCTATGAAAACGTGGGCCTGATGCAA CAGCAGAAAAGTTTCAGATGA PTPN12 H.sapiens ATGGAGCAAGTGGAGATCCTGAGGAAATTCATCCAGAGGGTCCAGGC SEQ CATGAAGAGTCCTGACCACAATGGGGAGGACAACTTCGCCCGGGACT ID TCATGCGGTTAAGAAGATTGTCTACCAAATATAGAACAGAAAAGATAT No. ATCCCACAGCCACTGGAGAAAAAGAAGAAAATGTTAAAAAGAACAGA 101 TACAAGGACATACTGCCATTTGATCACAGCCGAGTTAAATTGACATTA AAGACTCCTTCACAAGATTCAGACTATATCAATGCAAATTTTATAAAG GGCGTCTATGGGCCAAAAGCATATGTAGCAACTCAAGGACCTTTAGCA AATACAGTAATAGATTTTTGGAGGATGGTATGGGAGTATAATGTTGTG ATCATTGTAATGGCCTGCCGAGAATTTGAGATGGGAAGGAAAAAATG TGAGCGCTATTGGCCTTTGTATGGAGAAGACCCCATAACGTTTGCACC ATTTAAAATTTCTTGTGAGGATGAACAAGCAAGAACAGACTACTTCAT CAGGACACTCTTACTTGAATTTCAAAATGAATCTCGTAGGCTGTATCA GTTTCATTATGTGAACTGGCCAGACCATGATGTTCCTTCATCATTTGAT TCTATTCTGGACATGATAAGCTTAATGAGGAAATATCAAGAACATGAA GATGTTCCTATTTGTATTCATTGCAGTGCAGGCTGTGGAAGAACAGGT GCCATTTGTGCCATAGATTATACGTGGAATTTACTAAAAGCTGGGAAA ATACCAGAGGAATTTAATGTATTTAATTTAATACAAGAAATGAGAACA CAAAGGCATTCTGCAGTACAAACAAAGGAGCAATATGAACTTGTTCAT AGAGCTATTGCCCAACTGTTTGAAAAACAGCTACAACTATATGAAATT CATGGAGCTCAGAAAATTGCTGATGGAGTGAATGAAATTAACACTGA AAACATGGTCAGCTCCATAGAGCCTGAAAAACAAGATTCTCCTCCTCC AAAACCACCAAGGACCCGCAGTTGCCTTGTTGAAGGGGATGCTAAAG AAGAAATACTGCAGCCACCGGAACCTCATCCAGTGCCACCCATCTTGA CACCTTCTCCCCCTTCAGCTTTTCCAACAGTCACTACTGTGTGGCAGGA CAATGATAGATACCATCCAAAGCCAGTGTTGCAATGGTTTCATCAGAA CAACATTCAGCAGACCTCAACAGAAACTATAGTAAATCAACAGAACTT CCAGGGAAAAATGAATCAACAATTGAACAGA PTPN22 H.sapiens ATGGACCAAAGAGAAATTCTGCAGAAGTTCCTGGATGAGGCCCAAAG SEQ CAAGAAAATTACTAAAGAGGAGTTTGCCAATGAATTTCTGAAGCTGAA ID AAGGCAATCTACCAAGTACAAGGCAGACAAAACCTATCCTACAACTG No. TGGCTGAGAAGCCCAAGAATATCAAGAAAAACAGATATAAGGATATT 102 TTGCCCTATGATTATAGCCGGGTAGAACTATCCCTGATAACCTCTGAT GAGGATTCCAGCTACATCAATGCCAACTTCATTAAGGGAGTTTATGGA CCCAAGGCTTATATTGCCACCCAGGGTCCTTTATCTACAACCCTCCTGG ACTTCTGGAGGATGATTTGGGAATATAGTGTCCTTATCATTGTTATGGC ATGCATGGAGTATGAAATGGGAAAGAAAAAGTGTGAGCGCTACTGGG CTGAGCCAGGAGAGATGCAGCTGGAATTTGGCCCTTTCTCTGTATCCT GTGAAGCTGAAAAAAGGAAATCTGATTATATAATCAGGACTCTAAAA GTTAAGTTCAATAGTGAAACTCGAACTATCTACCAGTTTCATTACAAG AATTGGCCAGACCATGATGTACCTTCATCTATAGACCCTATTCTTGAGC TCATCTGGGATGTACGTTGTTACCAAGAGGATGACAGTGTTCCCATAT GCATTCACTGCAGTGCTGGCTGTGGAAGGACTGGTGTTATTTGTGCTA TTGATTATACATGGATGTTGCTAAAAGATGGGATAATTCCTGAGAACT TCAGTGTTTTCAGTTTGATCCGGGAAATGCGGACACAGAGGCCTTCAT TAGTTCAAACGCAGGAACAATATGAACTGGTCTACAATGCTGTATTAG AACTATTTAAGAGACAGATGGATGTTATCAGAGATAA GalK Escherichia ATGAGTCTGAAAGAAAAAACACAATCTCTGTTTGCCAACGCATTTGGC SEQ coli TACCCTGCCACTCACACCATTCAGGCGCCTGGCCGCGTGAATTTGATT ID GGTGAACACACCGACTACAACGACGGTTTCGTTCTGCCCTGCGCGATT No. GATTATCAAACCGTGATCAGTTGTGCACCACGCGATGACCGTAAAGTT 103 CGCGTGATGGCAGCCGATTATGAAAATCAGCTCGACGAGTTTTCCCTC GATGCGCCCATTGTCGCACATGAAAACTATCAATGGGCTAACTACGTT CGTGGCGTGGTGAAACATCTGCAACTGCGTAACAACAGCTTCGGCGGC GTGGACATGGTGATCAGCGGCAATGTGCCGCAGGGTGCCGGGTTAAG TTCTTCCGCTTCACTGGAAGTCGCGGTCGGAACCGTATTGCAGCAGCT TTATCATCTGCCGCTGGACGGCGCACAAATCGCGCTTAACGGTCAGGA AGCAGAAAACCAGTTTGTAGGCTGTAACTGCGGGATCATGGATCAGCT AATTTCCGCGCTCGGCAAGAAAGATCATGCCTTGCTGATCGATTGCCG CTCACTGGGGACCAAAGCAGTTTCCATGCCCAAAGGTGTGGCTGTCGT CATCATCAACAGTAACTTCAAACGTACCCTGGTTGGCAGCGAATACAA CACCCGTCGTGAACAGTGCGAAACCGGTGCGCGTTTCTTCCAGCAGCC AGCCCTGCGTGATGTCACCATTGAAGAGTTCAACGCTGTTGCGCATGA ACTGGACCCGATCGTGGCAAAACGCGTGCGTCATATACTGACTGAAAA CGCCCGCACCGTTGAAGCTGCCAGCGCGCTGGAGCAAGGCGACCTGA AACGTATGGGCGAGTTGATGGCGGAGTCTCATGCCTCTATGCGCGATG ATTTCGAAATCACCGTGCCGCAAATTGACACTCTGGTAGAAATCGTCA AAGCTGTGATTGGCGACAAAGGTGGCGTACGCATGACCGGCGGCGGA TTTGGCGGCTGTATCGTCGCGCTGATCCCGGAAGAGCTGGTGCCTGCC GTACAGCAAGCTGTCGCTGAACAATATGAAGCAAAAACAGGTATTAA AGAGACTTTTTACGTTTGTAAACCATCACAAGGAGCAGGACAGTGCTG A SacB Bacillus ATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACT SEQ subtilis ACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAAC ID GAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACG No. CCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATC 104 AAGTTCCTGAATTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAA AGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCA CTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAG ATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAG TCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTA AAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAA ACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATC CGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACA CTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAAC ATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAA ACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCA GGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGG CCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTA CCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCAC ATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAA ACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAA CGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAA CACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACG GCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTG ACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTC TTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAA AATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCT GTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGAC AAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCAAGCTT CCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCA TCCTTGAACAAGGACAATTAACAGTTAACAAATAA

ABBREVIATIONS

[0533] PTP IB, protein tyrosine phosphatase IB; TC-PTP, T-cell protein tyrosine phosphatase; S11P2, protein tyrosine phosphatase non-receptor type 11; BBR, 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide; TC5401, 2-[(Carbox-ycarbonyl)amino]-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid hydrochloride; AA, abietic acid; SCA, statistical coupling analysis. PTP1B1-435, protein tyrosine phosphatase 1B (full-length); SacB, levansucrase; GHS, ?-humulene synthase; ADS, amorphadiene synthase; ABS (or AgAs), abietadiene synthase; TXS, taxadiene synthase, PTPN5, protein tyrosine phosphatase non-receptor type 5; PTPN6, protein tyrosine phosphatase non-receptor type 6; PTPN1 1, protein tyrosine phosphatase non-receptor type 11; PTPN12, protein tyrosine phosphatase non-receptor type 12; PPTN22, protein tyrosine phosphatase non-receptor type 22; RpoZ, omega subunit of RNA polymerase; cI (or cI434), cI repressor protein from lambda phage; Kras (or p130cas), p130cas phosphotyrosine substrate; MidT, phosphotyrosine substrate from hamster polyoma virus; EGFR substrate, phosphotyrosine substrate from epidermal growth factor receptor; Src, Src kinase; CDC37, Hsp90 co-chaperone Cdc37; MBP, maltose-binding protein; LuxAB, bacterial luciferase modules A and B; SpecR, spectinomycin resistance gene; GGPPS, geranylgeranyl diphosphate synthase; P450 (or P450BM3) Cytochrome P450; LOV2, light-oxygen-voltage domain 2 from phototropin 1; BphP1, bacterial phytochrome; Galk, galatokinase.

EXAMPLES

[0534] The following examples are offered to illustrate various embodiments of the invention, but should not be viewed as limiting the scope of the invention.

[0535] Statistical Analysis of Kinetic Models. We evaluated four kinetic models of inhibition as described previously (19). In brief, we used an F-test to compare a two-parameter mixed model to several single-parameter models, and we used Akaike's Information Criterion (AIC, or Ai) to compare the single-parameter models to one another. Mixed models with p<0.05 are superior to all single-parameter models, and single-parameter models with Aj>10 are inferior to the reference (i.e., best fit) model.

[0536] Exemplary Estimation of IC50. We estimated the half maximal inhibitory concentration (IC50) of BBR by using kinetic models to estimate the concentration of inhibitor required to reduce initial rates of PTP-catalyzed hydrolysis of 20 mM of pNPP by 50%, and we used the MATLAB function nlparci to determine the confidence intervals on those estimates (19).

[0537] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in medicine, molecular biology, cell biology, genetics, statistics or related fields are intended to be within the scope of the following claims.