LIGHT-SWITCHABLE POLYPEPTIDE AND USES THEREOF

Abstract

The present invention relates to a light-switchable polypeptide. In particular, the present invention relates to a polypeptide comprising a light-responsive element, wherein the configuration (i.e. the configurational state) of the light-responsive element can be switched between a trans and cis isomer by irradiating the polypeptide with (a) particular wavelength(s) of light, and wherein the switch of said configuration alters the conformation and binding activity of said polypeptide to a ligand (e.g. molecule of interest). Also, the present invention comprises using said light-switchable polypeptide for isolating and/or purifying a molecule of interest. The present invention further provides an affinity matrix, an affinity chromatography column, and an affinity chromatography apparatus comprising the light-switchable polypeptide of the invention.

Claims

1. A polypeptide comprising a light-responsive element, wherein the light-responsive element can be switched between two isomers by irradiating the polypeptide with a particular wavelength of light, thereby altering the binding activity of the polypeptide to a ligand.

2-3. (canceled)

4. A method for isolating and/or purifying a molecule of interest, comprising: contacting a liquid phase comprising the molecule of interest with the polypeptide of claim 1, wherein the polypeptide is part of a solid phase, and wherein the light-responsive element is in a first configuration so that the polypeptide has high affinity to the molecule of interest; and (ii) irradiating the polypeptide with a wavelength that changes the light-responsive element to a second configuration so that the polypeptide has a decreased affinity to the molecule of interest as compared to the affinity of step (i); and (iii) eluting the molecule of interest from the solid phase.

5. The polypeptide of claim 1, wherein the polypeptide is streptavidin or a variant or mutein thereof comprising a light-responsive element.

6. The polypeptide of claim 1, wherein the polypeptide comprises or consists of (i) the amino acid sequence of SEQ ID NO: 2; (ii) the amino acid sequence of SEQ ID NO: 4; (iii) the amino acid sequence of SEQ ID NO: 6; (iv) the amino acid sequence of SEQ ID NO: 86; (v) the amino acid sequence of SEQ ID NO: 20, wherein the residue at position 12 of SEQ ID NO: 20 is replaced by a light-responsive element; (vi) the amino acid sequence of SEQ ID NO: 61, wherein the residue at position 13 of SEQ ID NO: 61 is replaced by a light-responsive element; or (vii) an amino acid sequence having at least 80% identity to the amino acid sequence according to any one of (i)-(vi) wherein irradiating the polypeptide results in a change in conformation or shape of a ligand-binding pocket of the polypeptide the polypeptide.

7. (canceled)

8. The polypeptide of claim 1, wherein the light-responsive element is in or in the vicinity of a ligand-binding pocket or site of the polypeptide or wherein the light-responsive element is involved in binding of a ligand to the polypeptide.

9. (canceled)

10. The polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 20, 61, or 86 or an amino acid sequence having at least 80% identity thereto and the light-responsive element is (i) at amino acid position 96 of any one of SEQ ID NOs: 2, 4, 8, and 10; (ii) at position 132 of any one of SEQ ID NOs: 6 and 12; (iii) at position 12 of SEQ ID NO: 20; (iv) at position 13 of any one of SEQ ID NOs: 61 and 86; (v) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 8 or 10, at the amino acid position that is homologous to amino acid position 96 of SEQ ID NO: 2, 4, 8 or 10, respectively; (vi) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 6 or 12, at the amino acid position that is homologous to amino acid position 132 of SEQ ID NO: 6 or 12, respectively; (vii) in an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 20, at the amino acid position that is homologous to amino acid position 12 of SEQ ID NO: 20; or (viii) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 61 and 86, at the amino acid position that is homologous to amino acid position 13 of SEQ ID NO: 61.

11. The polypeptide of claim 1, wherein the polypeptide has higher affinity to a ligand before being irradiated as compared to the polypeptide after it has been irradiated.

12. The polypeptide of claim 1, wherein the polypeptide has high affinity to a ligand before being irradiated and the polypeptide has low affinity to said ligand after it has been irradiated.

13. The polypeptide of claim 1, wherein the light-responsive element comprises an azo group or a light-switchable amino acid side chain.

14. (canceled)

15. The polypeptide of claim 1, wherein the light-responsive element comprises a non-natural amino acid, wherein two isomers of the non-natural amino acid can be switched with particular wavelengths of light.

16. The polypeptide of claim 1, wherein the light-responsive element comprises (i) 3-carboxyphenylazophenylalanine or a derivative thereof; or (ii) 4-carboxyphenylazophenylalanine or a derivative thereof.

17. The polypeptide of claim 1, wherein the isomers are a trans isomer and a cis isomer.

18. The polypeptide of claim 17, wherein the a trans isomer of 3-carboxyphenylazophenylalanine or 4-carboxyphenylazophenylalanine has an increased affinity to a ligand as compared to a cis isomer of 3-carboxyphenylazophenylalanine or 4-carboxyphenylazophenylalanine.

19. The polypeptide of claim 17, wherein at visible light having 405-470 nm, at least 70% of the polypeptide comprises a trans isomer of the light-responsive element or at ultraviolet (UV) light having 310 to 370 nm, at least 85% of the polypeptide comprises a cis isomer of the light-responsive element.

20.-21. (canceled)

22. A solid phase selected from the group consisting of a matrix, a hydrogel, a bead, a magnetic bead, a chip, a glass surface, a plastic surface, a gold surface, a silver surface, and a plate, wherein the solid phase comprises the polypeptide of claim 1.

23.-28. (canceled)

29. The polypeptide of claim 1, wherein the polypeptide binds to a ligand selected from the group consisting of a peptide, an oligopeptide, a polypeptide, a protein, an antibody or a fragment thereof, an immunoglobulin or a fragment thereof, an enzyme, a hormone, a cytokine, a complex, an oligonucleotide, a polynucleotide, a nucleic acid, a carbohydrate, a liposome, a nanoparticle, a cell, a biomacromolecule, a biomolecule, and a small molecule.

30. The polypeptide of claim 29, wherein the ligand comprises or consists of (i) the amino acid sequence of SEQ ID NO: 13; (ii) the amino acid sequence of SEQ ID NO: 14; or (iii) an amino acid sequence having at least 80% identity to SEQ ID NO: 13 or 14 and having affinity to streptavidin or its mutants or variants.

31. The polypeptide of claim 30, wherein the polypeptide is a streptavidin mutant comprising a tetramer of the protein having the amino acid sequence of SEQ ID NO: 7.

32.-44. (canceled)

45. An affinity matrix comprising the polypeptide of claim 1.

46. An affinity chromatography column comprising the affinity matrix of claim 45.

47.-53. (canceled)

Description

[0186] The Figures show:

[0187] FIG. 1: Principle of light-controlled affinity chromatography for protein purification.

[0188] The affinity column contains a chromatography matrix with an immobilized light-switchable binding protein (affinity molecule). A protein solution (e.g. a cell extract) is applied to the column and, once the protein of interest (e.g. carrying an affinity tag such as the Strep-tag II) has bound to the affinity matrix, contaminating proteins and biomolecules (possibly including host cell and/or buffer components of any kind) are washed away. By irradiation with mild UV light at 365 nm the conformation of the binding protein in the affinity matrix is changed in such a way as to lose binding activity towards the protein of interest and/or affinity tag, thus effecting instant elution (under constant buffer flow). To regenerate the column afterwards, green light >530 nm is applied, which relaxes the affinity matrix to the ground state.

[0189] FIG. 2: Synthesis of the photo-switchable non-natural amino acid 4-carboxyphenylazophenylalanine alias 4-[(4-carboxyphenyl)azo]-L-phenylalanine based on azo-benzene.

[0190] (A) Preparation of 4-[(4-carboxyphenyl)azo]-L-phenylalanine (Caf; 7) via Boc- or Fmoc-protected intermediates, also illustrating the reversible isomerization from the trans to the cis configuration triggered by light at different wavelengths. (B).sup.1H-NMR spectrum of 4-[(4-carboxyphenyl)azo]-L-phenylalanine (7) in D.sub.2O. (C).sup.13C-NMR spectrum of 4-[(4-carboxyphenyl)azo]-L-phenylalanine (7) in D.sub.2O.

[0191] FIG. 3: Synthesis of the photo-switchable non-natural amino acid 3-carboxyphenylazophenylalanine alias 4-[(3-carboxyphenyl)azo]-L-phenylalanine based on azo-benzene.

[0192] (A) Preparation of 4-[(3-carboxyphenyl)azo]-L-phenylalanine (11), also illustrating the reversible isomerization from the trans to the cis configuration triggered by light at different wavelengths.

[0193] (B) .sup.1H-NMR spectrum of 4-[(3-carboxyphenyl)azo]-L-phenylalanine (11) in D.sub.2O. (C) .sup.13C-NMR spectrum of 4-[(3-carboxyphenyl)azo]-L-phenylalanine (11) in D.sub.2O.

[0194] FIG. 4: Reversible photo-switching (isomerization) of Caf with alternating 365 nm (UV) versus 530 nm (green) LED photo-irradiation cycles.

[0195] (A) UV spectrum of the non-natural amino acid Caf in water (solid line: trans isomer; dotted line: cis isomer). (B) Reversible photo-switching between trans and cis configurations as visualized via changes in absorbance at approximately 340 nm (transition .fwdarw.*) over 3 cycles. High absorption at 335 nm indicates the trans configuration whereas low absorption at 335 nm indicates the cis configuration of Caf, cf. panel (A). (C-D) HPLC chromatograms of 7, absorption at =286 nm before irradiation (C), after irradiation with UV light (D) and after irradiation with green light (E). The chromatogram in panel (C) reveals essentially pure trans isomer; the chromatogram in panel (D) reveals mostly cis isomer, with the trans isomer as minor species; the chromatogram in panel (E) reveals mostly trans isomer, with the cis isomer as minor species.

[0196] FIG. 5: Structural and sequence overview of SAm1.sup.Caf variants.

[0197] (A) Crystal structure of the complex between streptavidin mutant 1 (SAm1, Strep-Tactin) and the Strep-tag II with highlighted residues V44, W108 and W120 (PDB entry 1KL3). All positions substituted with Caf were investigated for their potential to interfere with binding (reduce affinity) of the Strep-tag II in the cis configuration of the non-natural amino acid Caf but preserve binding in its (trans) ground state. Among these positions investigated for introduction of Caf as a light-responsive element, V44 and W120 are less preferred. (B) Nucleic and amino acid sequence of SAm1 with positions for Caf incorporation (in translation/suppression of an amber stop codon) highlighted.

[0198] FIG. 6: Expression, purification and refolding of SAm1.sup.Caf108.

[0199] (A) Plasmid map of pSBX8.CafRS#30d53 (SEQ ID NO: 55). (B) Purification and refolding of the recombinant core streptavidin mutant SAm1 carrying Caf at position 108. An SDS-PAGE (15%) gel stained with Coomassie brilliant blue is shown with samples from different stages during preparation of the recombinant protein. Lanes: 1, total E. coli protein before induction of gene expression; 2, total cell protein 12 h after induction; 3, protein solution after renaturation of the inclusion bodies and CEX purification; 4, same sample as in 3, but without heat treatment prior to SDS-PAGE. Under these conditions the core streptavidin tetramer remains intact (Bayer et al. 1990 Methods Enzymol. 184: 80-89). Thus the correctly folded state of the recombinant mutant streptavidin in the final preparation was confirmed whereas small amounts of monomeric (likely non-functional) streptavidin were still present after refolding (lane 4). Lane 5 shows the same sample as lane 4, but at lower concentration.

[0200] FIG. 7: Reversible binding of the PhoA/Strep-tag II fusion protein to streptavidin mutants/variants modified with a light-switchable amino acid in an ELISA.

[0201] (A) ELISA setup for screening streptavidin mutants having reversible binding activity toward the Strep-tag II peptide in response to UV light. (B) Screening for light-induced desorption of purified PhoA/Strep-tag II from SAm1 and its variants Caf44, Caf108, Caf120. All tested streptavidin mutants showed good affinity for the PhoA/Strep-tag II fusion protein, giving rise to comparable signals as obtained with SAm1 for those samples illuminated with visible light. In contrast, a clear decrease in remaining enzyme activity was observed after irradiation with UV light at 365 nm for the streptavidin variant SAm1.sup.Caf108. This indicates reduced affinity of the streptavidin variant SAm1.sup.Caf108 for PhoA carrying the Strep-tag II upon light-induced switching of Caf to the cis configuration.

[0202] FIG. 8: Light-induced desorption of PhoA/Strep-tag II from a functionalized affinity matrix.

[0203] (A) Flow profile observed for the chromatography column containing 20 L sepharose with immobilized SAm1.sup.Caf108. Irradiation with green LED light (530 nm) or mild UV light (365 nm) was performed as indicated. (B) Samples of each fraction (10 L) collected from the SAm1.sup.Caf108 column were analyzed by SDS-PAGE. Lanes: M, molecular size standard; L, loaded sample; FT, flow-trough; W, wash; E1-E3, elution fractions. (C) 15% SDS-PAGE of samples from the SAm1 column. (D) Quantification of PhoA/Strep-tag II fusion protein in the collected fractions (loaded sample, flow-through, wash, elution 1-3) via PhoA enzyme assay. In contrast to the unmodified streptavidin mutein (SAm1), the affinity column comprising SAm1.sup.Caf108 reveals light-dependent elution of the bound PhoA/Strep-tag II fusion protein.

[0204] FIG. 9: Structural and sequence overview of ProtL.sup.Caf variants.

[0205] (A) Crystal structure of the complex between the trastuzumab Fab fragment and the B1 domain of protein L with Caf337 as well as mutated residues Asn361 and Ser365 shown as sticks (UniProt accession code Q51918; this corresponds to positions 29, 53 and 57 in the PDB entry 4 HKZ). Position 337 is suitable for substitution with Caf with the goal of achieving a different affinity towards an immunoglobulin depending on the cis or trans configuration of the light-responsive non-natural amino acid. (B) Nucleic acid and amino acid sequence of the ProtL.sup.Caf-ABD fusion protein with position 337 for Caf incorporation (in translation/suppression of an amber stop codon) highlighted. Methionine (underlined) was added as a start codon in comparison to SEQ ID NO: 20.

[0206] FIG. 10: Expression and purification of the ProtL.sup.Caf337-ABD fusion protein

[0207] An SDS-PAGE (15%) gel stained with Coomassie brilliant blue shows samples from different stages during preparation of the recombinant protein. Lanes: 1, total E. coli protein before induction of gene expression; 2, total cell protein 12 h after induction; 3, insoluble fraction of the whole cell extract; 4, soluble supernatant of the whole cell extract; 5, elution fraction from HSA affinity chromatography; 6, ProtL.sup.Caf337-ABD after CEX purification.

[0208] FIG. 11: Reversible binding of an immunoglobulin to a ProtL.sup.Caf-ABD fusion protein modified with a light-switchable amino acid in an ELISA.

[0209] (A) Schematic ELISA setup for screening of ProtL.sup.Caf variants having reversible binding activity towards immunoglobulins in response to UV light. (B) Exemplary assay for light-induced desorption of a mouse anti-6His antibody (immunoglobulin) conjugated with alkaline phosphatase (AP) from protein L domain B1 and its variant Caf337 (both fused with the ABD and adsorbed to an HSA-coated microtiter plate). In its ground state, the tested Caf337 variant showed high affinity for the IgG (right, hollow circles), even though with lower signals than observed for the unmodified Protein L domain (left, hollow circles). In contrast, a clear decrease in remaining activity of bound Ig-AP conjugate was observed after irradiation with UV light at 365 nm (solid circles) only for the ProtL.sup.Caf337 variant, indicating that the light-induced formation of the cis isomer of Caf leads to specific dissociation between the light-switchable Protein L domain and the immunoglobulin (Ig).

[0210] Error bars indicate standard deviations from triplicate measurements. Curve fit of the ELISA data (Voss & Skerra 1997 Protein Eng 10:975-82) for the ProtL.sup.Caf337 in its ground state revealed a dissociation constant of approximately 140 nM for the complex with the anti-6His antibody, corresponding to a high affinity. The signal intensities observed after irradiation with UV light were too low to deduce a dissociation constant, indicating strong loss in affinity of the light-switchable polypeptide (hence, these data were fitted by a straight line).

[0211] The Examples illustrate the invention.

EXAMPLE 1: SYNTHESIS OF 4-[(4-CARBOXYPHENYL)AZO]-L-PHENYLALANINE (CAF)

[0212] The preparation of 4-[(4-carboxyphenyl)azo]-L-phenylalanine (Caf; 7) (herein also called 4-carboxyphenylazophenylalanine) was previously reported (Nakayama et al. 2005 Bioconjug. Chem. 16: 1360-1366). However, here a more convenient protocol for the synthesis of Caf which is illustrated in FIG. 2A is provided. Commercially available Fmoc- or Boc-protected 4-amino-L-phenylalanine (3 and 4) was reacted with 4-nitrosobenzoic acid (2), which was prepared from 4-aminobenzoic acid (1) by oxidation with oxone (2 KHSO.sub.5+KHSO.sub.4+K.sub.2SO.sub.4). The resulting diazo intermediate 5 was deprotected with piperidine, whereas the alternative intermediate 6 was deprotected with HCl in dioxane, in both cases yielding the desired amino acid 7.

Step 1: Synthesis of 4-Nitrosobenzoic Acid (2)

[0213] Compound 2 was prepared according to a published procedure (Priewisch & Ruck-Braun 2005 J. Org. Chem. 70: 2350-2352). 4-Aminobenzoic acid (15 g, 109 mmol) was suspended in 180 ml dichloromethane. A solution of oxone (134.5 g, 219 mmol) in 675 ml H.sub.2O was added and the mixture was stirred for 1.5 h at room temperature. The precipitate was filtered off, washed thoroughly with H.sub.2O, dried at air and then over P.sub.2O.sub.5. 4-Nitrosobenzoic acid (2) was obtained as a yellow solid (16 g, 106 mmol), containing a small amount of 4-nitrobenzoic acid, and was further used without purification.

[0214] .sup.1H NMR (400 MHz, DMSO-d6) =13.50 (s, 1H, COOH), 8.29-8.22 (m, 2H, aromat.), 8.05-8.00 (m, 2H, aromat.).

[0215] .sup.13C NMR (101 MHz, DMSO) =166.19 (CO), 165.00 (C aromat.), 136.53 (C aromat.), 131.02 (2 C aromat.), 120.62 (2 C aromat.).

[0216] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA in 30 min, flow rate 0.6 ml/min; t.sub.R=14.43 min.

Step 2a: Synthesis of N-Fmoc-4-[(4-carboxyphenyl)azo]-L-phenylalanine (5)

[0217] Compound 5 was prepared analogously to a published procedure (Priewisch & Ruck-Braun 2005 J. Org. Chem. 70: 2350-2352). 4-Nitrosobenzoic acid (2) (3 g, 19.9 mmol) was suspended in 320 ml DMSO/AcOH 1:1 (v/v) with ultrasonification, followed by addition of Fmoc-Phe(4-NH.sub.2)OH (3) (4 g, 9.94 mmol; Iris Biotech, Marktredwitz, Germany). The mixture was stirred for 2 d at room temperature. Then 700 ml H.sub.2O was added and the resulting precipitate was filtered, washed with H.sub.2O, dried at air and then over P.sub.2O.sub.5. The desired product 5 was obtained as a brown solid and further used without purification.

[0218] .sup.1H NMR (400 MHz, DMSO-d6) =13.16 (s, 2H, 2COOH), 8.17-8.13 (m, 2H, aromat.), 7.97-7.90 (m, 2H, aromat.), 7.88-7.81 (m, 5H, aromat., NH), 7.68-7.58 (m, 2H, aromat.), 7.56-7.48 (m, 2H, aromat.), 7.42-7.33 (m, 2H, aromat.), 7.33-7.23 (m, 2H, aromat.), 4.30 (ddd, J=10.6, 8.5, 4.5 Hz, 1H, C.sup.H), 4.25-4.19 (m, 2H, Fmoc-CH.sub.2), 4.19-4.12 (m, 1H, Fmoc-CH), 3.23 (dd, J=13.9, 4.4 Hz, 1H, C.sup.H), 3.01 (dd, J=13.8, 10.7 Hz, 1H, C.sup.H).

[0219] .sup.13C NMR (101 MHz, DMSO) =173.14 (CO), 166.75 (CO), 155.99 (CO), 154.37 (C aromat.), 150.69 (C aromat.), 143.78 (C aromat.), 143.73 (C aromat.), 142.83 (C aromat.), 140.71 (C aromat.), 140.69 (C aromat.), 132.71 (C aromat.), 131.03 (2C aromat.), 130.66 (2C aromat.), 130.35 (2C aromat.), 127.61 (2C aromat.), 127.05 (2C aromat.), 122.79 (2C aromat.), 122.47 (2C aromat.), 120.09 (2C aromat.), 65.64 (Fmoc-CH.sub.2), 55.19 (C.sup.), 46.61 (Fmoc-CH), 36.42 (C.sup.).

[0220] MS analysis: calc. [M-H.sup.+]=534.16706; found [M-H.sup.+]=534.15320.

[0221] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=21.16 min.

Step 2b: Synthesis of N-Boc-4-[(4-carboxyphenyl)azo]-L-phenylalanine (6)

[0222] Compound 6 was prepared according to a published procedure (Bose et al. 2006 J. Am. Chem. Soc. 128: 388-389) Boc-Phe(4-NH.sub.2)OH (4) (1 g, 3.6 mmol; Bachem, Bubendorf, Switzerland) was dissolved in 50 ml AcOH. After addition of 4-nitrosobenzoic acid (2) (0.8 g, 5.4 mmol) the mixture was stirred for 24 h. The solvent was removed at reduced pressure and the remaining material was dissolved in 100 ml each of 1 M HCl (aq.) and ethyl acetate. The aqueous phase was extracted four times with 50 ml ethyl acetate. The combined organic phases were washed once with brine and dried over MgSO.sub.4. After evaporation of the solvent 6 was obtained as a brown solid (638 mg, 1.54 mmol, 43%), which was further used without purification.

[0223] .sup.1H NMR (500 MHz, DMSO-d6) =8.14 (d, J=8.3 Hz, 2H, aromat.), 7.94 (d, J=8.4 Hz, 2H, aromat.), 7.85 (d, J=7.9 Hz, 2H, aromat.), 7.49 (d, J=8.1 Hz, 2H, aromat.), 7.11 (d, J=8.4 Hz, 1H, NH), 4.22-4.13 (m, 1H, C.sup.H), 3.15 (dd, J=13.9, 4.6 Hz, 1H, C.sup.H), 2.95 (dd, J=13.8, 10.2 Hz, 1H, C.sup.H), 1.31 (s, 9H, C(CH.sub.3).sub.3).

[0224] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=18.33 min.

Step 3a: Synthesis of 4-[(4-Carboxyphenyl)azo]-L-phenylalanine (7) (Fmoc Cleavage)

[0225] Compound 5 (5 g, 9.34 mmol) was dissolved in 40 ml DMF, then 10 ml piperidine was added dropwise and the mixture was stirred for 30 min at room temperature. Addition of 450 ml 0.5 M NaHCO.sub.3 (aq.) caused formation of a colorless precipitate, which was removed by filtration. The filtrate was acidified to pH 1-2 by addition of 6 M HCl (aq.). The precipitate was filtered off and dried at air, then over P.sub.2O.sub.5. Compound 7 was obtained as a brown solid (2.42 g, 7.72 mmol, 98% over 2 steps) which was used for biophysical and biochemical experiments described in Example 3 and 6 without further purification.

[0226] .sup.1H NMR (400 MHz, D.sub.2O) =7.86-7.80 (m, 2H, aromat.), 7.59-7.53 (m, 2H, aromat.), 7.53-7.47 (m, 2H, aromat.), 7.24-7.18 (m, 2H, aromat.), 3.42 (dd, J=7.5, 5.6 Hz, 1H, C.sup.H), 2.90 (dd, J=13.5, 5.6 Hz, 1H, C.sub.H), 2.73 (dd, J=13.4, 7.6 Hz, 1H, C.sup.H).

[0227] .sup.13C NMR (101 MHz, D.sub.2O) =181.94 (CO), 174.43 (CO), 153.16 (C aromat.), 150.42 (C aromat.), 142.79 (C aromat.), 138.45 (C aromat.), 130.23 (2C aromat.), 129.81 (2 C aromat.), 122.55 (2 C aromat.), 121.93 (2 C aromat.), 57.28 (V), 40.80 (C.sup.).

[0228] MS analysis: calc. [M-H.sup.+].sup.=312.09898; found [M-H.sup.+].sup.=312.09380.

[0229] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=10.7 min.

Step 3b: Synthesis of 4-[(4-Carboxyphenyl)azo]-L-phenylalanine (7) (Boc Cleavage)

[0230] Compound 6 (638 mg, 1.5 mmol) was dissolved in 20 ml of approx. 2 M HCl in dioxane and stirred over night at room temperature. The precipitate was filtered off, washed with diethyl ether and dried at vacuum. Compound 7 was obtained as a brown solid (236 mg, 0.67 mmol, 44%), which was used for biophysical and biochemical experiments described in Example 3 and 6 without further purification. Analytical data were in agreement with those described in Step 3a.

EXAMPLE 2: SYNTHESIS OF 4-[(3-CARBOXYPHENYL)AZO]-L-PHENYLALANINE (11)

[0231] 4-[(3-Carboxyphenyl)azo]-L-phenylalanine (11) (herein also called 3-carboxyphenylazophenylalanine) was synthesized in 3 steps as shown in FIG. 3A. Fmoc-protected 4-aminophenylalanine (3) was reacted with 3-nitrosobenzoic acid (9), which was prepared from 3-aminobenzoic acid (8) by oxidation with axone. Intermediate 10 was deprotected with piperidine to yield 4-[(3-carboxyphenyl)azo]-L-phenylalanine (11).

Step 1: Synthesis of 3-Nitrosobenzoic Acid (9)

[0232] Compound 9 was prepared according to a published procedure (Priewisch & Ruck-Braun 2005 J. Org. Chem. 70: 2350-2352). 3-Aminobenzoic acid (8) (5 g, 36.5 mmol) was suspended in 100 ml DCM. After addition of a solution of oxone (44.9 g, 73 mmol) in 400 ml H.sub.2O, the mixture was stirred for 1 h at room temperature. The precipitate was filtered off, washed thoroughly with H.sub.2O, and dried over P.sub.2O.sub.5. 3-Nitrosobenzoic acid (9) was obtained as a brown solid (4.1 g, 27 mmol, 76%), containing a small amount of 3-nitrobenzoic acid, and was further used without purification.

[0233] .sup.1H NMR (400 MHz, DMSO-d6) =13.52 (s, 1H, COOH), 8.41-8.35 (m, 1H, aromat.), 8.35-8.33 (m, 1H, aromat.), 8.19-8.11 (m, 1H, aromat.), 7.91-7.84 (m, 1H, aromat.).

[0234] .sup.13C NMR (101 MHz, DMSO) =166.08, 165.19, 136.26, 132.45, 130.47, 124.25, 120.98. Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=14.05 min.

Step 2: Synthesis of N-Fmoc-4-[(3-carboxyphenyl)azo]-L-phenylalanine (10)

[0235] Compound 10 was prepared analogously to a published procedure (Priewisch & Ruck-Braun 2005 J. Org. Chem. 70: 2350-2352). 3-Nitrosobenzoic acid (9) (378 mg, 2.5 mmol) was suspended in 40 ml DMSO/AcOH 1:1 with ultrasonification, followed by addition of Fmoc-Phe(4-NH.sub.2)OH (3) (500 mg, 1.24 mmol). The mixture was stirred for 2 d at room temperature and then 200 ml H.sub.2O was added. The resulting precipitate was filtered, washed with H.sub.2O, and dried over P.sub.2O.sub.5. Fmoc-protected amino acid 10 was obtained as a brown solid and further used without purification.

[0236] .sup.1H NMR (400 MHz, DMSO-d6) =13.15 (s, 2H, 2COOH), 8.38-8.33 (m, 1H, aromat.), 8.11 (dd, J=7.8, 1.8 Hz, 2H, aromat.), 7.93-7.79 (m, 5H, aromat., NH), 7.77-7.69 (m, 1H, aromat.), 7.63 (t, 2H, aromat.), 7.54-7.48 (m, 2H, aromat.), 7.43-7.33 (m, 2H, aromat.), 7.33-7.22 (m, 2H, aromat.), 4.28 (ddd, J=10.8, 8.5, 4.5 Hz, 1H, C.sup.H), 4.24-4.10 (m, 3H, Fmoc-CH, CH.sub.2), 3.26-3.17 (m, 1H, C.sup.H), 3.00 (dd, J=13.8, 10.7 Hz, 1H, C.sup.H).

[0237] .sup.13C NMR (101 MHz, DMSO) =173.11 (CO), 166.72 (CO), 155.96 (C aromat.), 151.94 (CO), 150.55 (C aromat.), 143.77 (2 C aromat.), 143.71 (2 C aromat.), 142.51 (C aromat.), 140.67 (C aromat.), 136.26 (C aromat.), 132.15 (C aromat.), 130.48 (C aromat.), 130.30 (2 C aromat.), 129.95 (C aromat.), 127.59 (C aromat.), 127.04 (2 C aromat.), 125.23 (C aromat.), 125.18 (C aromat.), 122.67 (2 C aromat.), 122.22 (C aromat.), 120.08 (2 C aromat.), 65.62 (Fmoc-CH2), 55.18 (Fmoc-CH), 46.57 (C.sup.), 36.36 (C.sup.).

[0238] MS analysis: calc. [M-H.sup.+].sup.=534.16706; found [M-H.sup.+].sup.=534.15493.

[0239] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck KgaA, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=21.6 min.

Step 3: Synthesis of 4-[(3-Carboxyphenyl)azo]-L-phenylalanine (11)

[0240] The Fmoc-protected amino acid 10 (650 mg, 1.21 mmol) was dissolved in 12 ml DMF. After dropwise addition of 3 ml piperidine the mixture was stirred for 30 min at room temperature. Addition of 35 ml 0.5 M NaOH caused formation of a colorless precipitate, which was removed by filtration. The filtrate was acidified to pH 1-2 using 6 M HCl (aq.). The resulting precipitate was removed by filtration and dried at air, then over P.sub.2O.sub.5. Amino acid 11 was obtained as a brown solid (361 mg, 1.15 mmol, 83% over 2 steps) and was used for biophysical experiments described in Example 3 without further purification.

[0241] .sup.1H NMR (400 MHz, D.sub.2O) =8.14-8.08 (m, 1H, aromat.), 7.94-7.89 (m, 1H, aromat.), 7.76-7.70 (m, 1H, aromat.), 7.67-7.60 (m, 2H, aromat.), 7.55-7.47 (m, 1H, aromat.), 7.34-7.27 (m, 2H, aromat.), 3.48 (dd, J=7.4, 5.6 Hz, 1H, C.sup.H), 2.97 (dd, J=13.5, 5.6 Hz, 1H, C.sup.H.sub.2), 2.81 (dd, J=13.5, 7.5 Hz, 1H, C.sup.H.sub.2).

[0242] .sup.13C NMR (101 MHz, D.sub.2O) =182.01 (CO), 174.34 (CO), 151.76 (C aromat.), 150.50 (C aromat.), 142.60 (C aromat.), 137.59 (C aromat.), 131.49 (C aromat.), 130.28 (2 C aromat.), 129.26 (C aromat.), 123.84 (C aromat.), 123.29 (C aromat.), 122.52 (2 C aromat.), 57.32)(C, 40.78 (C.sup.).

[0243] MS analysis: calc. [M-H.sup.+].sup.=312.09898; found [M-H.sup.+].sup.=312.09760.

[0244] Analytical HPLC: Column Purospher RP-8e 2503 mm (Merck, Darmstadt, Germany), gradient 10-100% ACN in water+0.1% TFA over 30 min, flow rate 0.6 ml/min; t.sub.R=11.3 min.

EXAMPLE 3: LIGHT-INDUCED ISOMERIZATION OF 4-[(4-CARBOXYPHENYL)AZO]-I-PHENYLALANINE (CAF)

Analysis by Spectroscopy

[0245] The UV-VIS absorption spectrum of azobenzene reveals two characteristic absorption bands corresponding to .fwdarw.* and n.fwdarw.* electronic transitions, which differ in amplitude and precise location of the absorption maximum () for the trans and cis configuration. The electronic transition .fwdarw.* is usually in the near UV region around 340 nm (Sension et al. 1993 J. Chem. Phys. 98: 6291-6315) whereas the electronic transition n.fwdarw.* is usually located in the visible (VIS) region around 420 nm and is due to the presence of unshared electron pairs of the nitrogen atoms (Naegele et al. 1997 Chem. Phys. Lett. 272: 489-495). To examine whether the synthesized non-natural amino acid Caf (7) can respond to photoswitching induced by UV light, the compound was subjected to alternating irradiation cycles. In a typical experiment, 0.5 ml of a 30 M aqueous solution was placed in a quartz cuvette with 1 cm optical pathlength. Then the sample was irradiated for 30 min from the top using a UV LED (NS355L-5RLO; Nitride Semiconductors, Tokushima, Japan) with 353 nm or a green LED (LL-504PGC2E-G5-2CC; Lucky Light Electronics, Hongkong, China) with 520 nm emitting wavelength. The change in intensity of the -* band at around 340 nm corresponding to the trans/cis isomerization (FIG. 4A) was monitored with a computer controlled photometer (Ultrospec 2100 pro, Amersham Biosciences). Closer examination revealed reproducible changes in absorbance at about 340 nm over 3 cycles, consistent with reversible photoswitching between the trans (high absorbance at 340 nm) and cis (low absorbance at 340 nm) configuration of the azo compound (FIG. 4B).

Analysis by HPLC

[0246] 500 l of a 60 M solution of Caf (7) in water was placed in a 1.5 ml HPLC vial (Screw neck vial N9, amber glass, 11.632 mm; Macherey Nagel, Duren, Germany) and irradiated with a UV LED (=353 nm, NS355L-5RLO; Nitride Semiconductors, Tokushima, Japan) for 30 min directly from the top. Before and after irradiation, a 20 l sample of the solution was withdrawn and analyzed by HPLC on a Purospher RP-8e 2503 mm column (Merck), applying a concentration gradient of 10-12% acetonitrile (ACN) in 50 mM NH.sub.4OAc buffer pH 8 over 10 min (flow rate 0.6 ml/min). Another sample was analyzed in the same manner after irradiation with green LED light (=520 nm, LL-504PGC2E-G5-2CC, Lucky Light Electronics, Hongkong, China). FIG. 4 shows the corresponding chromatograms with absorbance at =286 nm (wavelength at which trans-(7) and cis-(7) show the same molar extinction coefficient, allowing direct comparison of peak integrals). The chromatograms reveal that the cis and trans isomers of (7) can be separated by HPLC (cis-(7) t.sub.R=3.6 min, trans-(7) t.sub.R=4.6 min). Prior to irradiation in the ground state, only energetically favored trans-(7) occurs (FIG. 4C). Irradiation with UV light (365 nm) causes an increase in the proportion of cis-(7), here up to 86% (FIG. 4D), which can be reversed by irradiation with green light (=520 nm), thus recovering the ground state (FIG. 4E) via photochemical reisomerization. However, it should be taken into account that also during HPLC analysis reisomerization of cis-(7) to trans-(7) takes place, so the proportion of cis-(7) after irradiation with UV light might actually be higher than indicated by HPLC chromatograms. Thus, if the light-switchable polypeptide of the present invention is applied for an affinity chromatography procedure, and the trans configuration corresponds to the high affinity state whereas the cis configuration corresponds to the low affinity conformation, then the highest degree of binding and the highest degree of elution of the molecule of interest takes place at 430 nm and 330 nm, respectively. However, conventional light sources usually provide light having wavelengths that are around 530 nm (visible light) and 365 nm (UV light). Therefore, also light providing these wavelengths (i.e. around 530 nm and/or around 365 nm) may be used in accordance with the present invention.

EXAMPLE 4: SELECTION OF A PYLRS VARIANT SPECIFIC FOR 4-[(4-CARBOXYPHENYL)AZO]-L-PHENYLALANINE (CAF)

[0247] The biosynthesis of proteins containing a photo-switchable non-natural amino acid such as 4-[(4-carboxyphenyl)azo]-L-phenylalanine (Caf) opens the way to novel light-controllable biomolecular reagents for biophysical, structural or biochemical research as well as biotechnological and biopharmaceutical applications. To develop an orthogonal pair of suppressor tRNA and amino-acyl tRNA synthetase (aaRS) for the co-translational site-specific incorporation of Caf in a recombinant protein produced in E. coli, the pyrrolysyl-tRNA synthetase (PylRS) from the methanogenic archaeon Methanosarcina barkeri (Mb) (James et al. 2001 J. Biol. Chem. 276: 34252-34258) and its cognate tRNA.sup.Pyl that specifically recognizes and suppresses the amber stop codon (Fekner & Chan 2011 Curr. Opin. Chem. Biol. 15:387-91) were employed.

[0248] To select a mutant aaRS specific for the non-natural amino acid substrate Caf, a previously described one-plasmid system (Kuhn et al. 2010 J. Mol. Biol. 404: 70-87) encoding both the aaRS and the cognate tRNA was adapted to PylRS. The modified plasmid, pSBX8.101d58 (SEQ ID NO: 23), encodes a PylRS derived from Mb and the cognate suppressor tRNA.sup.Pyl (FIG. 5A). Cloned on the same plasmid, a chloramphenicol-resistance reporter gene equipped with an amber stop codon (cat.sup.UAG112; SEQ ID NO: 24) served to select highly active aaRS variants (conferring Cam resistance), and a fluorescent reporter gene equipped with another amber stop codon (eGFP.sup.UAG39; SEQ ID NO: 25) was used in conjunction with fluorescence-activated cell sorting (FACS) to screen for variants exhibiting the desired amino acid specificity. By applying alternating cycles of positive and negative FACS combined with dead/live selection on LB agar plates supplemented with Cam in the presence or in the absence of the foreign amino acid, respectively, a mutated aaRS (dubbed CafRS) with high specificity for Caf incorporation was selected.

[0249] The mutation Tyr349F has been described to increase the in vivo suppression activity of Mb PylRS for non-natural amino acids (Yanagisawa et al. 2008 Chem. Biol. 15: 1187-1197) and, therefore, this position was fixed to Phe in all libraries. The mutation was introduced into the PylRS wild-type gene (SEQ ID NO: 26) using the QuikChange site-directed mutagenesis kit (Agilent, Waldbronn, Germany) with a pair of suitable PCR primers (SEQ ID NO: 27 and 28), resulting in the variant PylRS#1 (SEQ ID NO: 29).

[0250] To evolve a mutant synthetase specific for the non-natural amino acid Caf, a first synthetase library (CafRS#0-R5) based on PylRS#1 was generated by fully randomizing five positions (M309, Asn311, Cys313, Met315 and Trp382) in the active site using NNS degenerate primers in a two-step assembly PCR approach. Site-directed saturation mutagenesis was carried out using the Q5 DNA polymerase PCR kit (New England Biolabs, Ipswich, Mass., USA) with the PylRS#1 gene (SEQ ID NO: 29) as template. First, two overlapping PCR fragments were prepared, each using a pair of forward and reverse primers (forward primer 1: SEQ ID NO: 30; forward primer 2: SEQ ID NO: 31; reverse primer 1: SEQ ID NO: 32; reverse primer 2: SEQ ID NO: 33). All primers were supplied by MWG Eurofins (Ebersberg, Germany).

[0251] The two randomization reactions were performed under the same conditions in a 50 L reaction mixture comprising 1 Q5 buffer, 200 M of each dNTP and 0.5 U Q5 DNA polymerase. The mixture was denatured for 10 s at 98 C., annealed for 30 s at 64 C., and a linear polymerase reaction was then performed for 30 s at 72 C. After 35 cycles, an enzymatic digest with DpnI was performed at 37 C. for 2 h to remove the bacterial template. Both amplified DNA fragments were purified via agarose gel purification using the Gel Extraction Kit (Qiagen, Hilden, Germany) and assembled in a second PCR reaction. To this end, 200 ng of both fragments were mixed in a 50 L Q5 DNA polymerase reaction mixture comprising 1 Q5 buffer, 200 M of each dNTP and 0.5 U Q5 DNA polymerase. The mixture was denatured for 10 s at 98 C., annealed for 30 s at 64 C., and a linear polymerase reaction was then performed for 30 s at 72 C. After 10 cycles the flanking primers (SEQ ID NOs: 30 and 34) were added, followed by 30 thermocycles of 10 s at 98 C., 30 s at 64 C. and 30 s at 72 C. with a final incubation at 72 C. for 5 min.

[0252] After agarose gel purification of the PCR product using the Qiagen Gel Extraction Kit reamplification was performed in 100 L Q5 DNA polymerase reaction mixture using primers SEQ ID NOs: 30 and 34 by applying the thermocycles described above. A pair of mutually non-compatible type IIS restriction sites (BsaI) in the flanking primers used in the preceding assembly step (SEQ ID NOs: 30 and 34) allowed unidirectional insertion of the central coding region into pSBX8.101d58 (SEQ ID NO: 23). After application of the Qiagen PCR purification Kit the resulting DNA fragment carrying random mutations in the targeted regions was doubly cut with BsaI, again purified using the Qiagen PCR purification Kit and cloned on the plasmid pSBX8.101d58. Transformation (Dower et al. 1988 Nucleic Acids Res. 16: 6127-6145) of electrocompetent E. coli NEB10beta cells (New England Biolabs) yielded a library of 310.sup.9 transformants (according to colony count of a sample fraction), which were plated on 10 square LB agar plates (114 cm.sup.2) supplemented with 100 mg/L ampicillin.

[0253] Colonies were scraped from the plates and resuspended in each 5 mL LB medium (Sambrook & Russell 2001 Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York, N.Y.), then combined and adjusted to a volume of 1 L with fresh medium. After incubation at 30 C. under shaking for 30 min, plasmid DNA was prepared from this pooled culture by means of the Qiagen Plasmid Midi Kit and subsequently used for transformation of electrocompetent E. coli BL21 (Studier & Moffatt 1986 J. Mol. Biol. 189: 113-130). Randomization of the targeted positions in the PylRS#1 gene cloned on pSBX8.101d58 was confirmed by DNA sequencing.

[0254] Directly after transformation (Dower et al. 1988 Nucleic Acids Res. 16: 6127-6145) of electrocompetent E. coli BL21 with the CafRS#0-R5 library prepared above, 4 mL of the transfected cell suspension were diluted in 50 mL LB medium supplemented with phosphate buffer (17 mM KH.sub.2PO.sub.4, 72 mM K.sub.2HPO.sub.4) and 1 mM Caf (100 mM stock solution in 300 mM NaOH). After incubation for 2 h at 37 C., cells were sedimented by centrifugation and washed with 10 mL fresh LB medium without additives. After another centrifugation step, cells were resuspended in 2 mL LB medium and plated on four square LB agar plates (114 cm.sup.2) supplemented with 100 mg/L ampicillin, 60 mg/L chloramphenicol and 1 mM Caf. Colonies obtained after incubation for 48 h at 37 C. were scraped from the plates and resuspended in 5 mL LB medium each, then combined and diluted into 1 L LB medium containing 100 mg/L ampicillin and grown at 37 C. to 0D.sub.550=0.4 in a 3 L shake flask. From this culture, triplicates of 2 mL cultures were transferred into plastic tubes and supplemented in parallel with or without 1 mM Caf, freshly dissolved as a 100 mM solution in 300 mM NaOH. Bacteria were grown under shaking at 37 C. for 30 min, then expression of eGFP was induced by addition of 200 ng/mL anhydrotetracycline (aTc; Acros Organics, Geel, Belgium) dissolved at 2 mg/mL in DMF, followed by shaking at 37 C. for another 9-12 h. 1 mL of each culture was centrifuged in a 1.5 mL Eppendorf tube for 3 min and the bacterial pellet was carefully resuspended by repeated pipetting with 1 mL filter-sterilised PBS (4 mM KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4, 115 mM NaCl). After washing twice according to this procedure, the bacteria were finally resuspended in the same volume of PBS.

[0255] Flow cytofluorimetric analysis as well as bacterial cell sorting were performed on a FACSAria instrument (BD Biosciences, Heidelberg, Germany) which was operated with filter-sterilised PBS as sheath fluid, using a 488 nm LASER for excitation and a 502 nm long-pass filter with a 530/30 band-pass filter for specific detection of eGFP fluorescence. After selecting intact bacterial cells via an appropriate FSC/SSC gate, the final sort gates for each population were dynamically set to select those cells belonging to the fraction of 1 to 5% of total cells with the highest eGFP signal intensities in the presence of Caf for positive selection cycles. For negative selection, cells with low eGFP signal, comparable to that of uninduced bacteria, were sorted. Bacteria were directly collected in LB medium supplemented with 100 mg/L ampicillin. For reamplification, the sorted cells were plated on LB agar containing 100 mg/L ampicillin and incubated at 37 C. over night. The lawn of colonies was collectively resuspended in LB medium as described further above. A 2 mL aliquot of this dense bacterial cell suspension was used to inoculate 100 mL freshly prepared LB medium supplemented with 100 mg/L ampicillin to be directly used for the next selection cycle.

[0256] To enrich CafRS variants with high fidelity and to eliminate those accepting any natural amino acid, two successive negative FACS selection steps were initially performed. Following five alternating FACS selection rounds of positive (i.e. with addition of 1 mM Caf) and negative selection (i.e. in the absence of Caf), a fluorescence response indicating specific incorporation of Caf into the reporter protein eGFP clearly developed. After the final positive selection cycle, bacteria were plated on LB agar and plasmid DNA was prepared from recovered cells by means of the Qiagen Plasmid Midi Kit. After transformation of calcium competent E. coli BL21 cells, followed by plating on LB agar supplemented with 100 mg/L ampicillin in a rectangular plastic dish (Nunc, Langenselbold, Germany), the resulting bacterial population was subjected to single-clone analysis in 96-well microcultures using a robotic platform as previously described in detail (Reichert et al. 2015 Protein Eng. Des. Sel. 28: 553-565). In this assay, 190 randomly chosen colonies were propagated and analyzed individually for eGFP fluorescence.

[0257] After incubation over night at 37 C., colonies were automatically picked and used to inoculate 100 L TB medium (Sambrook & Russell 2001 Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York, N.Y.) supplemented with 100 mg/L ampicillin in 96-well round bottom microtiter plates (Sarstedt, Nrnbrecht, Germany). The microtiter plates were sealed with a gas-permeable Breathseal 80/140 mm membrane (Greiner Bio-One, Frickenhausen, Germany) and incubated overnight at 37 C. to stationary phase under 300 rpm agitation using an orbital shaking Minitran incubator with 25 mm amplitude (Infors, Eisenbach, Germany). Then, fresh 1 mL cultures in TB medium containing 100 mg/L ampicillin were inoculated in Masterblock 2 mL V-shape deep well microtiter plates (Greiner Bio-One), each with 20 L of the pre-culture, and incubated for approximately 2 h at 37 C. to reach OD.sub.5500.5 as monitored with the Synergy 2 SLFA microplate reader (BioTek Instruments, Bad Friedrichshall, Germany). This inoculation step was done in duplicate using two equivalent 96 deep-well plates, one to be supplemented with 1 mM Caf and the other without the non-natural amino acid. After further shaking for 30 min the cells were induced with 200 ng/mL aTc (by adding 20 L from a 10 g/mL stock solution in LB medium). Bacterial growth was continued at 37 C. for 12 h; then, the cultures were centrifuged (3857g; 15 min) and resuspended in 1 mL PBS by repeated pipetting on the robotic platform. Washing in PBS was repeated once. Finally, eGFP.sup.Caf39 fluorescence of a 100 L aliquot was measured in the cell suspension using Maxisorb black 96-well assay plates (Nunc) under excitation at 395 nm, detecting emission at 510 nm with cutoff at 495 nm. Fluorescence readings of each well were normalised to 0D.sub.550 of the same cell suspension, diluted 1:5 (20 L aliquot plus 80 L PBS), in a 96-well Mikrotest plate F (Sarstedt). The normalised background fluorescence of two wells with cells harboring only empty pSBX8.100d backbone (encoding no eGFP) was averaged and subtracted from all other fluorescence readings. Final values were determined as fluorescence ratio aaRS.sup.+Caf/aaRS.sup.Caf for each clone.

[0258] The best clone in terms of efficiency and fidelity, dubbed CafRS#7 (SEQ ID NO: 35) showed already some increase in mean eGFP fluorescence, which indicated the need for randomization of further positions. Sequence analysis of CafRS#7 indicated three amino acid substitutions compared to PylRS#1 (Met309Gln, Asn311Ser and Cys313Gly).

[0259] CafRS#7 (SEQ ID NO: 35) was used as starting point for a second focused aaRS library (CafRS#7-R6; SEQ ID NO: 36) with six fully randomized positions (Ala267, Leu270, Tyr271, Leu274, Ile285 and Ile287). Two PCR fragments were generated using two sets of degenerate NNS-primers and assembled. The first PCR fragment was generated with a forward primer (SEQ ID NO: 30) and a NNS reverse primer (SEQ ID NO: 37) to introduce variations for the residues of interest, generating the upstream portion of the gene. The second PCR fragment was generated with another NNS-degenerate forward primer (SEQ ID NO: 38) and a reverse primer (SEQ ID NO: 34), having an overlap of the forward primer to the 3 end of the first PCR product, providing the downstream portion of the gene. These PCR fragments were generated according to the experimental procedure described above with the CafRS#7 gene serving as template. After agarose gel purification, 200 ng of each fragment was used in an assembly PCR reaction with primers for the 5 (SEQ ID NO: 30) and 3 ends (SEQ ID NO: 34) of the gene, also comprising the BsaI restriction sites. The library was cloned on pSBX8.101.d58, yielding 110.sup.10 transformants, and subjected to an initial dead/alive selection for viable colonies on LB agar plates supplemented with 100 mg/mL ampicillin as well as 30 mg/mL chloramphenicol and 1 mM Oaf, followed by 2 negative selection rounds using FACS. After five alternative FACS selections (three positive and two negative) bacterial cells were recovered on LB agar supplemented with 100 mg/L ampicillin, followed by single-clone analysis of 189 colonies in a 96-well microculture format as described above. Sequence analysis of the mutated aaRS gene cassettes revealed that the clone with the highest specific fluorescence ratio, dubbed CafRS#29 (SEQ ID NO: 39), carried four additional amino acid substitutions (Ala267Thr, Leu274Ala, Ile285Asn, Ile287Ser) as compared to CafRS#7.

[0260] Judged from the crystal structure of the Methanosarcina mazei (Mz) PylRS (PBD entry 2ZCE) and from the results of the two prior library screenings, two residues located at the entry (Gln309 and Ser311), which had already been targeted in the first library, and three residues located at the rear part of the active site (Ala274, Asn285 and Ser287), which had been targeted in the second library, appeared as promising candidates for constructing a third CafRS library. The library CafRS#29-R5 (SEQ ID NO: 40) based on CafRS#29 was generated again via assembly PCR using degenerate NNS primers.

[0261] Three PCR fragments were generated and assembled using a set of forward and reverse primers. The first PCR fragment was generated with a forward primer (SEQ ID NO: 30) and a NNS-degenerate reverse primer (SEQ ID NO: 41) to yield the randomized upstream portion of the gene. The second PCR fragment providing the middle part of the gene was generated with a set of two NNS-primers (SEQ ID NO: 38 and SEQ ID NO: 42) having an overlap with the 3 end of the first and the 5 end of the third PCR fragment. The third PCR fragment providing the downstream portion of the gene was generated with an NNS forward primer (SEQ ID NO: 31) and a reverse primer (SEQ ID NO: 34). The PCR fragments were generated and assembled according to the experimental procedure described above with the CafRS#29 gene serving as template. The gene library was digested with the restriction enzyme BsaI, gel purified, and ligated with the pSBX8.101d58 vector, after digestion with BsaI, to yield the CafRS#29-R5 library (SEQ ID NO: 40). 10 g of the ligation products were then electroporated into E. coli NEB10beta cells. Electroporated cells were recovered and plated on LB agar plates with 100 mg/mL ampicillin, yielding 110.sup.10 independent transformants. Selection from the CafRS#20-R5 library followed the procedure described for the selections from the first and the second CafRS-library. The finally selected mutant synthetase, CafRS#30 (SEQ ID NO: 43), carries in total 7 amino acid substitutions compared with wild-type Mb PylRS (Ala276Thr, Leu274Ser, Ile285Ser, IIle287Val, Asn311Val, Met315Gly and Tyr349Phe).

EXAMPLE 5: GENERATION OF SAM1.SUP.CAF .VARIANTS

[0262] For a proof of concept, the streptavidin mutant 1, SAm1 (also called Strep-Tactin) (Voss & Skerra 1997 Protein Eng. 10:975-82) (SEQ ID NOs: 7 and 8), was modified with Caf at either position V44, W108 or W120. To this end, an amber stop codon (TAG) was introduced into the coding region at each of these sequence positions by site-directed mutagenesis using the plasmid pSAm1 (SEQ ID NO: 44) as template together with the QuikChange site-directed mutagenesis kit and a suitable pair of forward and reverse PCR primers: SEQ ID NO: 45 and 46 resulting in SAm1.sup.UAG44 (SEQ ID NO: 47), SEQ ID NO: 48 and 49 resulting in SAm1.sup.UAG108 (SEQ ID NOs: 1 and 2) and SEQ ID NO: 50 and 51 resulting in SAm1.sup.UAG120 (SEQ ID NO: 52) (FIG. 5B). After transformation of calcium-competent E. coli XL1-blue cells, plasmid preparation (Plasmid Miniprep Kit, Qiagen) and sequencing (Mix2Seq, MWG Eurofins, Ebersberg, Germany), the SAm1 variants were subcloned via XbaI and HindIII on the vector pSBX8.CafRS#30d58 (SEQ ID NO: 53), yielding pSBX8.CafRS#30d47 (V44TAG; SEQ ID NO: 54), pSBX8.CafRS#30d53 (W108TAG; SEQ ID NO: 55) and pSBX8.CafRS#30d51 (W120TAG; SEQ ID NO: 56), respectively.

[0263] All positions substituted with Caf were intended to disturb binding of the Strep-tag II if the side chain adopts the cis configuration (i.e., after illumination at 340 or 365 nm) but preserve binding activity in the trans configuration (FIG. 5A). Position Val44 is located on the N-terminal side of the flexible loop region comprising positions 44-53. Caf isomerization was supposed to change the loop conformation. Position Trp108 is located at the bottom of the binding pocket for biotin and, therefore, cis-Caf was supposed to clash with neighboring side chains. Position Trp120 is located at the top of the binding site extending from a neighboring tetramer subunit, thus changing the overall geometry upon isomerization of Caf into the cis state.

EXAMPLE 6: EXPRESSION AND PURIFICATION OF SAM1 VARIANTS

[0264] Both SAm1 (SEQ ID NOs: 7 and 8) and the SAm1.sup.Caf variants were produced as cytoplasmic inclusion bodies in E. coli, solubilized, refolded, purified by anion-exchange chromatography (AEX) and analyzed by SDS-PAGE.

[0265] A single colony of E. coli BL21 transformed with plasmid pSBX8.CafRS#30d53 coding for SAm1.sup.Caf108 (SEQ ID NOs: 1 and 2) (FIG. 6A) was used for inoculating 50 mL LB medium supplemented with 100 mg/L ampicillin. After incubation overnight at 30 C. the 20 mL culture was transferred to 2 L LB medium in a baffled shake flask, again supplemented with 100 mg/L ampicillin as well as phosphate buffer (17 mM KH.sub.2PO.sub.4, 72 mM K.sub.2HPO.sub.4) and 1 mM Caf (from a 100 mM stock solution in 300 mM NaOH). The culture was incubated at 37 C. to OD.sub.550=0.5. Then, SAm1.sup.Caf108 gene expression (under control of the tet.sup.o/o; Skerra 1994 Gene 151: 131-135) was induced with 200 ng/mL aTc and growth was continued at 37 C. for 12 h. The CafRS gene was under the control of E. coli proS promotor and proM terminator. Cells were harvested by centrifugation (10,000g, 20 min, 4 C.) and washed twice with 100 mL 100 mM Na-borate pH 9.0, 150 mM NaCl to remove precipitated Caf. The bacteria were resuspended in 3 mL per mg wet weight of cold 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA and disrupted in 3 runs, using a French Pressure homogenizer (SLM Aminco, Urbana, Ill., USA). The homogenate was centrifuged (20.000 g, 30 min, 4 C.) to sediment the streptavidin inclusion bodies. After washing the protein pellet twice with 50 mM Tris-HCl pH 8.0, 2 M urea, 2% v/v Triton X-100 (3 mL/g cell wet weight) to remove impurities, followed by a washing step with 50 mM Tris-HCl to deplete residual Triton X-100. The inclusion bodies were dissolved in 8 M urea pH 2.5 (3 mL/g cell wet weight). After centrifugation (20.000 g, 30 min, 4 C.), the cleared supernatant was subjected to refolding, which was accomplished by rapid dilution. The unfolded protein was pipetted dropwise into a 25-fold volume of 50 mM Tris-HCl pH 8.0 at 4 C. using a Pasteur pipette. The mixture was incubated over night at 4 C., cleared by centrifugation (10.000g, 20 min, 4 C.) and purified by AEX on a 6 mL Resource Q column (GE Healthcare, Freiburg, Germany) equilibrated with 20 mM Tris-HCl pH 8.0. Protein fractions eluted in a linear salt concentration gradient of 0-500 mM NaCl at 80 mM NaCl in a pure state as analyzed by SDS-PAGE (Fling & Gregerson 1986 Anal. Biochem. 155: 83-88) using staining with Coomassie brilliant blue R-250 (FIG. 6B).

EXAMPLE 7: PREPARATION OF THE ALKALINE PHOSPHATASE/STREP-TAG II FUSION PROTEIN

[0266] Preparative protein expression of the PhoA/Strep-tag II fusion protein using E. coli JM83 transformed with the plasmid pASK75-PhoA-strepII (SEQ ID NO: 57) was accomplished in 2 L LB medium supplemented with 100 mg/mL ampicillin essentially as described by Voss & Skerra 1997 Protein Eng. 10: 975-982. Cultures were grown at 22 C. to OD.sub.550=0.5, then phoA gene expression was induced by addition of 200 ng/mL aTc. Incubation was continued at 22 C. for 4 h. Cells were harvested via centrifugation, resuspended in 20 mL ice-cold periplasmic fractionation buffer (0.5 M sucrose, 2 mg/mL polymyxcin B sulfate and 100 mM Tris-HCl, pH 8.0) containing 100 g/mL lysozyme and incubated for 30 min on ice. Due to the presence of metal ions in the active site of the enzyme, periplasmic protein preparation was carried out in the presence of 2 mg/mL polymyxin B sulfate instead of EDTA. The spheroplasts were removed by repeated centrifugation (Skerra & Schmidt 2000 Methods Enzymol. 326: 271-304) and the supernatant was recovered as periplasmic cell fraction. The PhoA/Strep-tag II fusion protein was purified from the periplasmic cell fraction by streptavidin affinity chromatography, using StrepTactin Sepharose (IBA, Gttingen, Germany) and D-desthiobiotin for elution according to a published procedure (Schmidt & Skerra 2007 Nat. Protoc. 2: 1528-1535). To avoid loss of metal ions in the active site of PhoA, EDTA was omitted from the chromatography buffer (150 mM NaCl, 100 mM Tris-HCl pH 8.0). Finally, the PhoA/Strep-tag II fusion protein was dialyzed twice against 2 L buffer (1 mM ZnSO.sub.4, 5 mM MgCl.sub.2, 100 mM Tris-HCl, pH 8.0) for removal of D-desthiobiotin prior to ELISA measurements or binding experiments with Caf-modified streptavidin variants immobilized on a chromatography matrix.

EXAMPLE 8: DETECTION OF REVERSIBLE BINDING FOR THE PHOA/STREP-TAG II FUSION PROTEIN IN AN ELISA

[0267] The light-induced reversible binding of streptavidin mutants carrying the light-switchable amino acid Caf at certain positions was first tested in an ELISA (enzyme-linked immunosorbent assay) using the purified PhoA/Strep-tag II fusion enzyme as a model ligand (FIG. 7).

[0268] ELISA was performed at ambient temperature in 96-well microtiter plates (Nunc, Langenselbold, Germany). Each well was coated over night with 100 L biotinylated bovine serum albumin (BSA) in PBS (4 mM KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4, 115 mM NaCl) at a concentration of 1 mg/mL (FIG. 7 A). Biotinylation of 2 mL BSA (10 mg/mL in PBS) was conducted using 20 molar excess of biotin NHS ester. After incubation for 2 h at room temperature, the reaction was quenched by addition of 2 mL 100 mM NaCl, 100 mM Tris-HCl pH 8.0 and purified using a PD-10 desalting column (GE Healthcare) equilibrated with the same buffer. The wells were blocked with 3% w/v BSA, 0.5% v/v Tween in PBS for 2.5 h and washed three times with PBS-Tween. 100 L of the SAm1 or its Caf-variants were applied at 100 g/mL in PBS to effect immobilization via complex formation of the pre-adsorbed biotin-BSA. After incubation for 1 h, the wells were washed three times with PBS-Tween. Then, 100 L of PhoA/Strep-tag II in 1 mM ZnSO.sub.4, 5 mM MgCl.sub.2: 100 mM Tris-HCl pH 8.0 was applied to each well. After incubation for 1 h, the liquid was removed and the wells were washed twice with PBS-Tween and twice with PBS. Between each of these washing steps, the microtiter plate was illuminated with UV light at a wavelength of 365 nm (UV hand lamp, NU-6 KL, Benda Laborgerte, Wiesloch, Germany; FIG. 7A, lower panel) with 2 mm distance, or with visible light (day light; FIG. 7A, upper panel), for 5 min, whereas buffer exchange was performed in the dark. Finally, 100 L 0.5 mg/mL p-nitrophenyl phosphate in 1 mM ZnSO.sub.4, 5 mM MgCl.sub.2, 1 M Tris-HCl, pH 8.0 was added to each well and remaining enzymatic PhoA activity was measured as the change in light absorption at 410 nm using a Synergy 2 SLFA microplate reader.

[0269] As a result, it appeared that all tested streptavidin mutants showed good affinity for the PhoA/Strep-tag II fusion protein, giving rise to comparable signals as obtained with SAm1 for those samples illuminated with visible light (FIG. 7B). In contrast, a clear decrease in remaining enzyme activity was observed after irradiation with UV light at 365 nm for the streptavidin variant SAm1.sup.Caf108. SAm1 as well as the mutants SAm1.sup.Caf44 and SAm1.sup.Caf120 showed no or much less signal decrease, respectively, under these circumstances. Hence, the streptavidin mutant SAm1.sup.Caf108 shows light-inducible (light-switchable) reversible binding of a target protein equipped with an affinity tag.

EXAMPLE 9: TEST OF A LIGHT-CONTROLLABLE AFFINITY MATRIX

[0270] Purified SAm1 or its Caf-variants, encoded on the corresponding derivative of vector pSBX8CAFRS#30 (see Examples 4 and 5), was coupled to NHS-activated Sepharose 4B (Pharmacia, Stockholm, Sweden) at 5 mg protein per mL of swollen gel as described (Schmidt & Skerra 1994 J. Chromatogr. A 676: 337-345). To this end, NHS-activated CH-Sepharose 4B was swollen and washed in ice-cold 1 mM HCl as recommended by the manufacturer. The supernatant was drained and the gel was mixed with twice its volume of a 2.5 mg/mL solution of the streptavidin variant which had been dialyzed against 100 mM NaHCO.sub.3 pH 8.0, 500 mM NaCl. After 2 h of gentle shaking at room temperature the supernatant was decanted and the gel was mixed with 5 volumes of 100 mM Tris-HCl pH 8.0 to achieve blocking of residual activated groups, followed by shaking overnight at 4 C.

[0271] A UV-transparent column was packed in a glass capillary (0.7 mm inner diameter) with 20 L of the chromatography matrix from above, each for Sam1.sup.Caf44, SAm1.sup.Caf108, SAm1.sup.Caf120 and SAm1, respectively. At first, the column was equilibrated twice with 2 mL running buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl) at a constant flow rate of 12 mL/h using a syringe pump (kdScientific, Holliston, Mass., USA), once under UV irradiation at 365 nm (UV hand lamp, NU-6 KL, Benda Laborgerte, Wiesloch, Germany) and once under irradiation using an LED light table (FG-08, Nippon Genetics, Dren, Germany) with an emitting wavelength of >530 nm (FIG. 8A). Then 25 L of the purified PhoA/Strep-tag II fusion protein with a concentration of 0.1 mg/mL in 100 mM Tris-HCl pH 8.0, 100 mM NaCl was applied and the column was washed with 2 mL running buffer while unbound protein was collected in the flow through fraction. Sample application and washing steps were conducted under irradiation with visible light using the LED light table. Subsequently, elution of bound protein was triggered by irradiation with UV light at 365 nm using the UV hand lamp. At first, buffer flow was stopped for 10 min while applying UV light. Then the flow rate was set to 12 mL/h again and three elution fractions (25 L each) were collected. The protein band visible on the Coomassie-stained gel corresponding to the PhoA/Strep-tag II fusion protein in the elution fractions of the chromatography matrix based on SAm1.sup.Caf108 indicates that the protein was specifically eluted by irradiation at 365 nm (FIG. 8B). No band was observed in case of streptavidin (FIG. 80) or its variants SAm1.sup.Caf44 and SAm1.sup.Caf120 (data not shown).

[0272] To increase the detection limit of affinity-purified protein in the elution fractions, PhoA enzyme activity was measured. Therefore, 10 L of each fraction (loaded sample, flow-through, washing and elution fractions 1-3) were applied to single wells of a 96-well plate (Nunc). 90 L 0.5 mg/mL p-nitrophenyl phosphate in 1 mM ZnSO.sub.4, 5 mM MgCl.sub.2, 1 M Tris-HCl, pH 8.0 was added. After incubation for 30 min at RT the enzymatic activity was determined by measuring time-dependent absorbance at 410 nm using a Synergy 2 SLFA microplate reader. In line with the SDS-PAGE analysis, the elution fractions of the chromatography matrix based on SAm1.sup.Caf108 showed the highest protein concentration (enzyme activity) eluted under UV irradiation (FIG. 8D).

EXAMPLE 10: GENERATION OF PROTL.SUP.CAF.-ABD VARIANTS

[0273] Protein L is a surface protein originally found in cell wall of Finegoldia magna (formerly known as Peptostreptococcus magnus) with a high affinity and specificity to immunoglobulins (Igs) from many mammalian species, most notably IgGs, and therefore has gained use for antibody purification (Rodrigo et al., 2015 Antibodies 4:259-277). While other IgG binding proteins like protein A and protein G from Staphylococcus aureus and group G Streptococci bind to the Fc region of Igs, protein L binds to the kappa light chain variable region without interfering with the antigen binding site. Natural protein L (UniProt accession number Q51918) essentially comprises the following domains (in analogy to Kaster et al. 1992 J. Biol. Chem. 267: 12820-12825): signal peptide (1-26); three protein G-related albumin-binding domains (77-116; 129-177; 190-238); four homologous B1 domains (254-317; 326-389; 399-436; 474-538); two C-repeats (610-660; 668-722) and a transmembrane region (969-991).

[0274] To engineer a light-switchable affinity matrix for the purification of antibodies as well as fragments or related formats (such as antibody fusion proteins, bispecific antibodies and the like), a recombinant protein L comprising a single domain without non-essential domains was designed. The codon optimized protein L domain B1 (herein referred to as ProtL; SEQ ID NO: 20) was fused to a human albumin-binding domain (ABD; SEQ ID NO: 59) derived from protein G via a short linker sequence. The protein L-ABD fusion protein (ProtL-ABD; SEQ ID NO: 61) was modified with Caf at either of the positions 337, 347, 360, 364, 368 or 369 (referring to the numbering scheme in UniProt accession number Q51918). The positions 337, 347, 360, 364, 368 and 369 correspond to positions 13, 23, 36, 40, 44, and 45, respectively, of SEQ ID NO: 61.

[0275] To this end, an amber stop codon (TAG) was introduced (via substitution of the original amino acid codon) into the coding region at each of these sequence positions by site-directed mutagenesis using the plasmid pASK75-ProtL-ABD (SEQ ID NO: 62) as template with the help of the QuikChange site-directed mutagenesis kit and a suitable pair of forward and reverse primers: SEQ ID NO: 63 and 66 for ProtL.sup.UAG337-ABD (SEQ ID NO: 67), SEQ ID NO: 68 and 69 for ProtL.sup.UAG347-ABD (SEQ ID NO: 72), SEQ ID NO: 73 and 74 for ProtL.sup.UAG360-ABD (SEQ ID NO: 75), SEQ ID NO: 76 and 77 for ProtL.sup.UAG364-ABD (SEQ ID NO: 78), SEQ ID NO: 79 and 80 for ProtL.sup.UAG368-ABD (SEQ ID NO: 81) and SEQ ID NO: 82 and 83 for ProtL.sup.UAG369-ABD (SEQ ID NO: 84) (FIG. 9).

[0276] After transformation of calcium-competent E. coli XL1-blue (Bullock et al., 1987 Biotechniques 5:376-378) cells, plasmid preparation and sequencing, the unmodified ProtL-ABD and the ProtL.sup.UAG-ABD variants were subcloned via XbaI and HindIII restriction sites on the vector pSBX8.CafRS#30d58 (SEQ ID NO: 53), yielding the plasmids pSBX8.CafRS#30d70 (no amber-stop codon), pSBX8.CafRS#30d71 (337TAG), pSBX8.CafRS#30d72 (347TAG), pSBX8.CafRS#30d73 (360TAG), pSBX8.CafRS#30d74 (364TAG), pSBX8.CafRS#30d75 (368TAG) and pSBX8.CafRS#30d76 (369TAG), respectively.

[0277] Positions 337 and 347 substituted with Caf were intended to disturb binding of Ig if the side chain adopts the cis configuration (i.e., after illumination at about 340 or about 365 nm) but retain binding activity in the trans configuration. Positions 360, 364, 368 and 369 substituted with Caf were intended to disturb Ig binding if the side chain adopts the trans configuration (i.e., after illumination >420 nm) but retain binding activity in the cis configuration. After isomerization the Caf side chain was supposed to clash with neighboring side chains within protein L (thus altering the conformation of its binding site) and/or the Ig ligand (thus changing the geometry of the protein/protein interface) and hence disturb binding.

[0278] To provide sufficient space for the large Caf side chain without sterical overlap (particularly in the extended trans configuration) within the binding interface of protein L and to preserve IgG binding activity, additional amino acid exchanges were introduced into the mutated ProtL-ABD as appropriate. For example, the mutation Tyr361Ala was introduced into the coding region of ProtL.sup.Caf347-ABD using the QuikChange site-directed mutagenesis kit and the forward and reverse primers SEQ ID NO: 70 and 73. The two additional mutations Tyr361Asn and Leu365Ser were simultaneous introduced into ProtL.sup.Caf337-ABD using the primers SEQ ID NO: 65 and 68. These positions 361 and 365 correspond to positions 37 and 41, respectively, of SEQ ID NO: 61 and 86.

EXAMPLE 11: EXPRESSION AND PURIFICATION OF PROTL.SUP.CAF.-ABD VARIANTS

[0279] ProtL (SEQ ID NO: 60) and the ProtL.sup.Caf variants (SEQ ID NOs: 69, 74, 77, 80, 83 and 86) were produced as ABD-fusion proteins in the cytoplasm of E. coli and purified by human serum albumin (HSA) affinity chromatography and anion-exchange chromatography (AEX).

[0280] For example, a single colony of E. coli MG1655 (Guyer et al., 1981 Cold Spring Harb Symp Quant Biol 45:135-40) transformed with plasmid pSBX8.CafRS#30d71, coding for ProtL.sup.Caf337-ABD (SEQ ID NO: 85), was used for inoculating 50 mL LB medium supplemented with 100 mg/L ampicillin. After incubation overnight at 30 C., 20 mL of the culture was transferred to 2 L LB medium supplemented with 100 mg/L ampicillin as well as phosphate buffer (17 mM KH.sub.2PO.sub.4, 72 mM K.sub.2HPO.sub.4) and 1 mM Caf (from a 100 mM stock solution in 300 mM NaOH) in a baffled shake flask. The culture was incubated at 37 C. to OD.sub.550=0.5 under agitation. Then, ProtL.sup.Caf337-ABD gene expression (under control of the tet.sup.o/o) was induced with 200 ng/mL aTc and growth was continued at 37 C. for 12-16 h. The CafRS gene was under the constitutive control of the E. coli proS promotor in combination with the proM terminator. Cells were harvested by centrifugation (10,000g, 20 min, 4 C.), resuspended in 3 mL per g wet weight of cold 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA and disrupted using a French Pressure homogenizer. The homogenate was centrifuged (20.000 g, 30 min, 4 C.) to sediment the cell debris, and the cleared supernatant was subjected to affinity chromatography using a HSA affinity column.

[0281] The HSA affinity matrix was prepared using NHS-activated Sepharose 4B (GE Healthcare, Freiburg, Germany) according to a published protocol (Schmidt & Skerra 1994 J. Chromatogr. A 676: 337-345). To this end, NHS-activated CH-Sepharose 4B was first swollen and washed in ice-cold 1 mM HCl as recommended by the manufacturer. The supernatant was drained and the gel was mixed with twice its volume of a 5 mg/mL solution of recombinant HSA produced in rice (Sigma-Aldrich, St. Louis, Mo., USA) in 100 mM NaHCO.sub.3 pH 8.0, 500 mM NaCl. After 2 h of gentle shaking at room temperature the supernatant was decanted and the gel was mixed with 5 volumes of 100 mM Tris-HCl pH 8.0 followed by shaking overnight at 4 C. in order to block residual activated groups. The HSA affinity matrix was packed into a 2 ml column housing connected to an KTA Purifier chromatography system.

[0282] After equilibration of the HSA column with running buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl) the cleared supernatant from E. coli containing ProL.sup.Caf337-ABD was loaded onto the column. Then, the column was washed with five volumes (10 mL) of running buffer and the bound protein was eluted with 150 mM glycine-HCl pH 2.8, 100 mM NaCl. Peak fractions were collected into neutralization buffer (100 l of 1 M Tris-HCl pH 9.0 per ml fraction), such that the final pH of the fractions became approximately neutral. Pooled fractions were immediately dialyzed against 20 mM Tris-HCl pH 8.0 at 4 C. over night. ProtL.sup.Caf337-ABD was further purified by AEX on a 1 mL Resource Q column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 8.0. Protein fractions were eluted in a linear salt concentration gradient of 0-200 mM NaCl at 100 mM NaCl in a pure state as analyzed by SDS-PAGE (Fling & Gregerson 1986 Anal. Biochem. 155: 83-88) as visualized by staining with Coomassie brilliant blue R-250 (FIG. 10). Other Caf variants as well as the unmodified ProtL-ABD fusion protein were prepared in the same manner.

EXAMPLE 12: DETECTION OF REVERSIBLE BINDING FOR THE PROTL.SUP.CAF.-ABD FUSION PROTEIN IN AN ELISA

[0283] The light-induced reversible binding of ProtL.sup.Caf-ABD mutants carrying the light-switchable amino acid Caf at certain positions was tested in an ELISA using a mouse anti-6His antibody alkaline phosphatase (AP) conjugate (Arigo Biolaboratories, Hsinchu City, Taiwan) as a model Ig ligand (FIG. 11 A). ELISA was performed at ambient temperature in a 96-well Maxisorb microtiter plate (Nunc, Langenselbold, Germany).

[0284] To this end, each well was first coated with 50 l of recombinant HSA produced in rice (Sigma-Aldrich) at a concentration of 10 g/ml in PBS (4 mM KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4, 115 mM NaCl) for 1 h at room temperature. Then, the wells were blocked with 200 ml Roti-Block (Carl Roth, Karlsruhe, Germany) diluted 1:10 in ddH.sub.2O for 1 h and washed three times with PBS containing 0.1% v/v Tween 20 (PBS/T). After that, the purified ProtL.sup.Caf-ABD fusion protein from Example 11 was applied in a dilution series in PBS/T and incubated for 1 h to effect complex formation between the ABD moiety and the pre-adsorbed HSA. The wells were then washed three times with PBS/T and incubated with 50 l of a 1:1000 dilution in PBS/T of the aforementioned mouse anti-6His Ig-AP conjugate.

[0285] After 1 h the microtiter plate was protected from daylight and illuminated with UV light at a wavelength of 365 nm (UV hand lamp NU-6 KL) with 2 mm distance for 5 min. All subsequent washing steps were performed in the dark. The microtiter plate was washed twice with PBS/T and twice with PBS, and then the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/mL in 5 mM MgCl.sub.2, 1 M Tris-HCl pH 8.0) as chromogenic substrate to quantify the remaining bound phosphatase reporter enzyme. After 5 min at 25 C., the absorbance at 405 nm was measured using a SpectraMax 250 microtiter plate reader (Molecular Devices, Sunnyvale, Calif., USA).

[0286] As result, the ProtL.sup.Caf337 variant (SEQ ID NO: 86) illuminated with visible light showed affinity for the IgG, even though with a lower signal than observed for ProtL without Caf (FIG. 11 B). In contrast, a clear decrease in enzyme activity was observed after irradiation with UV light at 365 nm for the ProtL.sup.Caf337 variant, whereas the unmodified ProtL-ABD fusion protein did not reveal any change in binding activity under the different illumination conditions. The mutants ProtL.sup.Caf347, ProtL.sup.Caf360, ProtL.sup.Caf364, ProtL.sup.Caf368 and ProtL.sup.Caf369 showed much less signal decrease under these circumstances. Hence, ProtL.sup.Caf337 shows light-switchable reversible binding of an IgG.

[0287] These experiments demonstrate that a chromatography matrix carrying an immobilized binding protein (engineered streptavidin or protein L) with the non-natural amino acid Caf incorporated at a suitable position in the polypeptide sequence can be used for the reversible binding and light-driven elution of a target protein (here equipped with and without an affinity tag) under typical conditions of an affinity chromatography, but without the need for application of a competing ligand or buffer shift.

[0288] The present invention refers to the following nucleotide and amino acid sequences:

TABLE-US-00003 SEQIDNO:1:Nucleicacidsequenceof Strep-TactincomprisingCaf. ThecodonofCafisinboldfaceandunderlined. ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTT CATCGTGACCGCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGG CGCGTGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGC GCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTG GAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGT ACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGACCTCC GGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACAC CTTCACCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:2:AminoacidsequenceofStrep-Tactin comprisingCaf. ThepositionofCafisinboldfaceandunderlined. MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThr PheIleValThrAlaGlyAlaAspGlyAlaLeuThrGlyThrTyrVal ThrAlaArgGlyAsnAlaGluSerArgTyrValLeuThrGlyArgTyr AspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThr ValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnCaf LeuLeuThrSerGlyThrThrGluAlaAsnAlaTrpLysSerThrLeu ValGlyHisAspThrPheThrLysValLysProSerAlaAlaSer

[0289] In the following, for illustration purposes, the amino acid sequence of Strep-Tactin comprising Caf (SEQ ID NO: 2) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 1). The position of Caf is in bold face and underlined.

TABLE-US-00004 102030405060 ++++++ 1ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC60 MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr 708090100110120 ++++++ 61GCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGGCGCGTGGCAACGCCGAGAGC120 AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrValThrAlaArgGlyAsnAlaGluSer 130140150160170180 ++++++ 121CGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCC180 ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla 190200210220230240 ++++++ 181CTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGG240 LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp 250260270280290300 ++++++ 241AGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGACCTCC300 SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnCafLeuLeuThrSer 310320330340350360 ++++++ 301GGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAG360 GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys 370380 ++ 361GTGAAGCCGTCCGCCGCCTCCTAA384 ValLysProSerAlaAlaSerEnd SEQIDNO:3:NucleicacidsequenceofcorestreptavidincomprisingCaf. ThecodonofCafisinboldfaceandunderlined. ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC GCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGA GCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACC GCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCAC GTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGA CCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTC ACCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:4:AminoacidsequenceofcorestreptavidincomprisingCaf. ThepositionofCafisinbddfaceandundedined. MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSer ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnCafLeuLeuThrSer GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys ValLysProSerAlaAlaSer

[0290] In the following, for illustration purposes, the amino acid sequence of core streptavidin comprising Caf (SEQ ID NO: 4) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 3). The position of Caf is in bold face and underlined.

TABLE-US-00005 102030405060 ++++++ 1ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC60 MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr 708090100110120 ++++++ 61GCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGC120 AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSer 130140150160170180 ++++++ 121CGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCC180 ArgTyrValLeuThrGlyArgTyrAspSerAlaProGlyThrAspGlySerGlyThrAla 190200210220230240 ++++++ 181CTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGG240 LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp 250260270280290300 ++++++ 241AGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGACCTCC300 SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnCafLeuLeuThrSer 310320330340350360 ++++++ 301GGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAG360 GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys 370380 ++ 361GTGAAGCCGTCCGCCGCCTCCTAA384 ValLysProSerAlaAlaSerEnd SEQIDNO:5:Nucleicacidsequenceofunprocessedstreptavidin (i.e.pre-streptavidin)comprisingCaf. ThecodonofCafisinboldfaceandunderlined. ATGCGCAAGATCGTCGTTGCAGCCATCGCCGTTTCCCTGACCACGGTCTCGATTACGGCC AGCGCTTCGGCAGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGG CATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCG ACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTC CTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTG GACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCC AGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGACCTCCGGCACC ACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAA GCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGC TCGACGCCGTTCAGCAGTAG SEQIDNO:6:Aminoacidsequenceofunprocessedstreptavidin (i.e.pre-streptavidin)comprisingCaf. Thesignalsequencewhichdirectssecretionofstreptavidinis underlined.ThepositionofCafisinboldfaceandunderlined. MetArgLvsIleValValAlaAlaIleAlaValSerLeuThrThrValSerIleThrAla SerAlaSerAlaAspProSerLysAspSerLysAlaGlnValSerAlaAlaGluAlaGly IleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThrAlaGlyAlaAsp GlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSerArgTyrValLeu ThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThr Va1AlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrpSerGlyGinTyr ValGlyGlyAlaGluAlaArgIleAsnThrGlnCafLeuLeuThrSerGlyThrThrGlu AlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLysValLysProSer AlaAlaSerIleAspAlaAlaLysLysAlaGlyValAsnAsnGlyAsnProLeuAspAla ValGlnGln

[0291] In the following, for illustration purposes, the amino acid sequence of unprocessed streptavidin (i.e. pre-streptavidin) comprising Caf (SEQ ID NO: 6) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 5). The signal sequence which directs secretion of streptavidin is underlined. The position of Caf is in bold face and underlined. The sequence of core streptavidin begins with Glu.sup.25 and ends with Ser.sup.163.

TABLE-US-00006 102030405060 ++++++ 1ATGCGCAAGATCGTCGTTGCAGCCATCGCCGTTTCCCTGACCACGGTCTCGATTACGGCC60 MetArgLysIlevalValAlaAlaIleAlaValSerLeuThrThrValSerIleThrAla 708090100110120 ++++++ 61AGCGCTTCGGCAGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGC120 SerAlaSerAlaAspProSerLysAspSerLysAlaGlnValSerAlaAlaGluAlaGly 14 130140150160170180 ++++++ 121ATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGAC180 IleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThrAlaGlyAlaAsp 190200210220230240 ++++++ 181GGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTG240 GlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSerArgTyrValLeu 250260270280290300 ++++++ 241ACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACG300 ThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThr 310320330340350360 ++++++ 301GTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTAC360 ValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrpSerGlyGlnTyr 370380390400410420 ++++++ 361GTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTAGCTGCTGACCTCCGGCACCACCGAG420 ValGlyGlyAlaGluAlaArgIleAsnThrGlnCafLeuLeuThrSerGlyThrThrGlu 430440450460470480 ++++++ 421GCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCC480 AlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLysValLysProSer 490500510520530540 ++++++ 481GCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCC540 AlaAlaSerIleAspAlaAlaLysLysAlaGlyValAsnAsnGlyAsnProLeuAspAla 163 550 + 541GTTCAGCAGTAG552 ValGlnGlnEnd SEQIDNO:7:Nucleicacidsequenceofstreptactin. ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC GCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGGCGCGTGGCAACGCCGAGAG CCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCG CCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGT GGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGAC CTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCA CCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:8:AminoacidsequenceofStrep-Tactin. Trp96isinboldfaceandunderlined. MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrValThrAlaArgGlyAsnAlaGluSer ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSer GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys ValLysProSerAlaAlaSer

[0292] In the following, for illustration purposes, the amino acid sequence of streptactin (SEQ ID NO: 8) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 7). The position of Trp is in bold face and underlined.

TABLE-US-00007 102030405060 ++++++ 1ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC60 MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr 14 708090100110120 ++++++ 61GCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGGCGCGTGGCAACGCCGAGAGC120 AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrValThrAlaArgGlyAsnAlaGluSer 130140150160170180 ++++++ 121CGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCC180 ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla 190200210220230240 ++++++ 181CTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGG240 LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp 250260270280290300 ++++++ 241AGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCC300 SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSer 310320330340350360 ++++++ 301GGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAG360 GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys 370380 ++ 361GTGAAGCCGTCCGCCGCCTCCTAA384 ValLysProSerAlaAlaSerEnd 139 SEQIDNO:9:Nucleicacidsequenceofcorestreptavidin. ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC GCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGA GCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACC GCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCAC GTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTG ACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTT CACCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:10:Aminoacidsequenceofcorestreptavidin(residues 2-127correspondtoresidues38-163inUniProtdatabaseentryP22629; residue1isastartmethionine).Trp96isinboldfaceandunderlined. MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSer ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSer GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys ValLysProSerAlaAlaSer

[0293] In the following, for illustration purposes, the amino acid sequence of core streptavidin (SEQ ID NO: 10) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 9). The position of Trp96 is in bold face and underlined.

TABLE-US-00008 102030405060 ++++++ 1ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC60 MetGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThr 708090100110120 ++++++ 61GCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGC120 AlaGlyAlaAspGlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSer 130140150160170180 ++++++ 121CGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCC180 ArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAla 190200210220230240 ++++++ 181CTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGG240 LeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrp 250260270280290300 ++++++ 241AGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCC300 SerGlyGlnTyrValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSer 310320330340350360 ++++++ 301GGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAG360 GlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLys 370380 ++ 361GTGAAGCCGTCCGCCGCCTCCTAA384 ValLysProSerAlaAlaSerEnd SEQIDNO:11:Nucleicacidsequenceofunprocessedstreptavidin (pre-streptavidin). ATGCGCAAGATCGTCGTTGCAGCCATCGCCGTTTCCCTGACCACGGTCTCGATTACGGCC AGCGCTTCGGCAGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGG CATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCG ACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTC CTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTG GACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCC AGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACC ACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAA GCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGC TCGACGCCGTTCAGCAGTAG SEQIDNO:12:Aminoacidsequenceofpre-streptavidin. Trp132isinboldfaceandunderlined. MetArgLysIleValValAlaAlaIleAlaValSerLeuThrThrValSerIleThrAla SerAlaSerAlaAspProSerLysAspSerLysAlaGlnValSerAlaAlaGluAlaGly IleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThrAlaGlyAlaAsp GlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSerArgTyrValLeu ThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThr ValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrpSerGlyGlnTyr ValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSerGlyThrThrGlu AlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLysValLysProSer AlaAlaSerIleAspAlaAlaLysLysAlaGlyValAsnAsnGlyAsnProLeuAspAla VaiGlnGln

[0294] In the following, for illustration purposes, the amino acid sequence of pre-streptavidin (SEQ ID NO: 12) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 11). The position of Trp132 is in bold face and underlined.

TABLE-US-00009 102030405060 ++++++ 1ATGCGCAAGATCGTCGTTGCAGCCATCGCCGTTTCCCTGACCACGGTCTCGATTACGGCC60 MetArgLysIleValValAlaAlaIleAlaValSerLeuThrThrValSerIleThrAla 708090100110120 ++++++ 61AGCGCTTCGGCAGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGC120 SerAlaSerAlaAspProSerLysAspSerLysAlaGlnValSerAlaAlaGluAlaGly 130140150160170180 ++++++ 121ATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGAC180 IleThrGlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThrAlaGlyAlaAsp 190200210220230240 ++++++ 181GGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTG240 GlyAlaLeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSerArgTyrValLeu 250260270280290300 ++++++ 241ACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACG300 ThrGlyArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThr 310320330340350360 ++++++ 301GTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTAC360 ValAlaTrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrpSerGlyGlnTyr 370380390400410420 ++++++ 361GTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAG420 ValGlyGlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSerGlyThrThrGlu 430440450460470480 ++++++ 421GCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCC480 AlaAsnAlaTrpLysSerThrLeuValGlyHisAspThrPheThrLysValLysProSer 490500510520530540 ++++++ 481GCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCC540 AlaAlaSerIleAspAlaAlaLysLysAlaGlyValAsnAsnGlyAsnProLeuAspAla 550 + 541GTTCAGCAGTAG552 ValGlnGlnEnd SEQIDNO:13:AminoacidsequenceofStrep-tag AWRHPQFGG SEQIDNO:14:AminoacidsequenceofStrep-tagII WSHPQFEK SEQIDNO:15Aminoacidsequenceofthemyc-tag(correspondingtoresidues410-419 inUniProtdatabaseentryP01106). EQKLISEEDL SEQIDNO:16:AminoacidsequenceofthedomainZofproteinA(correspondingto residues212-269inUniProtdatabaseentryP38507).SuitablepositionsforCaf incorporationPhe5,Gln9,Phe13,Tyr14,Glu25,Gln26,Arg27,Asn28Ala29,Phe30,Ile31, Gln32,Lys35,Asp36,Asp37,Gln40,Asn43,Leu45,Glu47,Leu51,Asn52showninbold faceandunderlined. VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQIDNO:17AminoacidsequenceoftheC1domainofproteinG(correspondingto residues303-357inUniProtdatabaseentryP19909).SuitablepositionsforCaf incorporationareLys3,Ile5,Thr10,Thr16,Val28,Tyr32,Asp35showninboldfaceand underlined. TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE SEQIDNO:18AminoacidsequenceoftheC2domainofproteinG(correspondingto residues373-427inUniProtdatabaseentryP19909).SuitablepositionsforCaf incorporationareLys3,Val5,Thr10,Thr16,Val28,Tyr32,Asp35showninboldfaceand underlined. TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE SEQIDNO:19AminoacidsequenceoftheC3domainofproteinG(correspondingto residues443-497inUniProtdatabaseentryP19909).SuitablepositionsforCaf incorporationareLys3,Val5,Thr10,Thr16,Ala28,Tyr32,Asp35showninboldfaceand underlined. TYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE SEQIDNO:20AminoacidsequenceofthedomainB1ofproteinL(correspondingto residues326-389inUniProtdatabaseentryQ51918).SuitablepositionsforCaf incorporationareThr5(330),Asn9(334),Ile(336),Phe12(337),Lys16(341),Phe22 (347)Phe26(351),Lys32(357),Ala35(360),Leu39(364),Glu43(368),Asn44(369)Tyr47 (372)showninboldfaceandunderlined. KEEVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFAG SEQIDNO:21Aminoacidsequenceoftheheavychainoftheanti-myc-tagmonoclonal antibodyclone9E10(correspondingtoresidues20-470inGenBankdatabaseentry CAN87018).SuitablepositionsforCafincorporationaretheresiduescorrespondingto Tyr76,Phe121,Tyr122,Tyr123,Tyr124,Tyr128,Tyr129andTyr130ofGenBankdatabase entryCAN87018,whichareshowninboldfaceandunderlined.Morespecifically,sincethe sequencebelowstartswithresidue20ofGenBankdatabaseentryCAN87018,thepositions whichcorrespondtoTyr76,Phe121,Tyr122,Tyr123,Tyr124,Tyr128,Tyr129andTyr130of GenBankdatabaseentryCAN87018arethepositionsTyr57,Phe102,Tyr103,Tyr104,Tyr105, Tyr109,Tyr110andTyr111,respectively,inthesequencebelow. EVHLVESGGDLVKPGGSLKLSCAASGFTFSHYGMSWVRQTPDKRLEWVATIGSRGTYTHYPD SVKGRFTISRDNDKNALYLQMNSLKSEDTAMYYCARRSEFYYYGNTYYYSAMDYWGQGASVT VSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPK DVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQD WPNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDI TVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTE KSLSHSPGK SEQIDNO:22Aminoacidsequenceofthelightchainoftheanti-myc-tagmonoclonal antibodyclone9E10(correspondingtoresidues21-238inGenBankdatabaseentry CAN87019). DIVLTQSPASLAVSLGQRATISCRASESVDNYGFSFMNWFQQKPGQPPKLLIYAISNRGSGVPA RFSGSGSGTDFSLNIHPVEEDDPAMYFCQQTKEVPWTFGGGTKLEIKRADAAPTVSIFPPSSE QLTSGGASVVCFLNNLYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC SEQIDNO:23:NucleicacidsequenceofpSBX8.101d58 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGATAACGAGGGCAAAAAATGTCTAAAGGTGAAGAACTTTTCACTGGAGTT GTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGA GGGTGAAGGTGATGCAACATAGGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAA CTACCTGTTCCATGGCCAACACTTGTCACTACTTTGACTTATGGTGTTCAATGCTTTTCAAG ATACCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTA CAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGA AACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGA CAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCG TTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCA GACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGGGACC ACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTGTA CCAAAGCGCTTGGAGCCACCCGCAGTTCGAAAAATAATAAGCTTGACCTGTGAAGTGAAAA ATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGCGGCAGTACG CCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGGGCCCTGAAATTCAGGACCCTTT CTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAGCTCTCGGAACGGCTTGCACGT TTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTCGAGACCAAAACCTGCACCAAT CCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACCGGACCAACAACTGCGCTACTC AGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCATAAACCATGCAGCTATCACCAA CAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTAATCAGTGCTTCCAGATTTTCA CGGGTACAACCGCTACCCATCTGACAAAAGTTCACCATTGTAAATTCTTCCAGGTGTTCTTT ACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAGATTTTGATAGGACCAGGCAGA ATACGATCCAGTTTCCGCAGGTAGTTATACAGTGTCGGTGCCAGCATAGGACGCAGACAC AGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGTATCATTGTTAATGCCCATACG TTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTTCCAGAAAACCGCGATCCACG AAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCTTCGCGGTCGTTGGTATATAA GCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTTCCAGTTCACGAAACGGTTTT GCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCTTCAACACGATCTAACTGAGA CCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTGCTATTCGGGGTGCTTTTTGC CGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTGCGCTAACGCTATTTTCCAGA GGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTAACTTTAGGTGCGCTCACAA CACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAGAAAATTGTTAATATCTTCAT CACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGATGACGAAATGCGCGTGCGGT ACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCACAGGCCATTTCAATATAGATTT TGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTGCCGGTACGGCTCATCCACAG ACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCATATCGTACCTCCTTAAATTTC TAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGAAATGTCAAAAGAGAAGGGCG TGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGCGTCGGTGCCGATTTCGGCCT ATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGC AGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGATTTAGAGTCCATTCGATCTAC ATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAAGTTTTTTATGTTGAGAATATT TTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCATGCAGGTGGCACTTTTCGG GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTC CAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGG GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTT AATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAG CAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTG AGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTT TTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAG TACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTT TTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGGGCATTT TACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGG TGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTA AATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAGGAATAGAGGATATGGAG AAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGA GGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCC TTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCC CGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATA TGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCT CTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCG TGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTC AGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCT TCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCT GGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGCTTCACTAGTTTAAAAGG ATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC SEQIDNO:24:Nucleicacidsequenceofcat.sup.UAG119 ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACA TTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTA CGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATT CTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTG GTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTC ATCGCTCTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATATATTCGCAAGAT GTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTT CGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGAC AACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGA TGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCT TAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAA SEQIDNO:25:NucleicacidsequenceofeGFP.sup.UAG39 ATGTCTAAAGGTGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGA TGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAGGGAAAA CTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCAC TACTTTGACTTATGGTGTTCAATGCTTTTCAAGATACCCGGATCATATGAAACGGCATGACT TTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGAC GGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCG AGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTT CAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATA CTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGC CCTTTCGAAAGATCCCAACGAAAAGAGGGACCACATGGTCCTTCTTGAGTTTGTAACAGCT GCTGGGATTACACATGGCATGGATGAACTGTACCAA SEQIDNO:26:NucleicacidsequenceofwtPylRS ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGGCACCGACACTGTATAACTACCTGCGGAAACTGGATCGTATTCTGCCTGGTCCTAT CAAAATCTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACAATGGTGAACTTTTGTCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTATGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTG SEQIDNO:27: TAGCTGCATGGTTTTTGGTGATACCCTGG SEQIDNO:28: CCAGGGTATCACCAAAAACCATGCAGCTA SEQIDNO:29:NucleicacidsequenceofPylRS#1(Y349F) ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGGCACCGACACTGTATAACTACCTGCGGAAACTGGATCGTATTCTGCCTGGTCCTAT CAAAATCTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACAATGGTGAACTTTTGTCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTG SEQIDNO:30: GAGCTTAACCCGGTCTCAGTTAGATCG SEQIDNO:31: GGTAGCGGTTGTACCCGTG SEQIDNO:32: GGGTACAACCGCTACCSNNCTGSNNAAASNNCACSNNTGTAATTCTTCCAGGTGTT SEQIDNO:33: CTTTCAGCAGACGTTCGAGACCAAAACCTGCACCAATSNNGGGTTTATCAATACCCCATTC SEQIDNO:34: CTTTCAGCAGACGTTCGAGAC SEQIDNO:35:NucleicacidsequenceofCafRS#7 ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGGCACCGACACTGTATAACTACCTGCGGAAACTGGATCGTATTCTGCCTGGTCCTAT CAAAATCTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACACAGGTGTCCTTTGGCCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTG SEQIDNO:36:NucleicacidsequenceofCafRS#7-R6 ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGNNNCCGACANNSNNSAACTACNNNCGGAAACTGGATCGTATTCTGCCTGGTCCTN NSAAANNSTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGA AGAATTTACACAGGTGTCCTTTGGCCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAA GCACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTG CATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTT GTTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGT TTTGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCC GTTCCGAGAGCTACTATAATGGTATTAGCACCAACCTG SEQIDNO:37: GGCAGAATACGATCCAGTTTCCGSNNGTAGTTSNNSNNTGTCGGSNNCAGCATAGGACGC AGACAC SEQIDNO:38: CGGAAACTGGATCGTATTCTGCCTGGTCCTNNSAAANNSTTTGAAGTTGGTCCGTGCTATC GT SEQIDNO:39:NucleicacidsequenceofCafRS#29 ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGACCCCGACATTGTTCAACTACGCGCGGAAACTGGATCGTATTCTGCCTGGTCCTAA CAAGAGCTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACACAGGTGTCCTTTGGCCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTGTAATGAGCTCAGAGAGGGTCCTGAT TTTCAGGGCCCTTTTTTTACGTGGTATTGTATAAAATGGATAAACCAAAGGCGTACTGCCGC GCAGTAGCGGTAAACGGCAGACAAAAAAAATGTCGCACAGTG SEQIDNO:40:NucleicacidsequenceofCafRS#29-R5 ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGACCCCGACATTGTTCAACTACNNSCGGAAACTGGATCGTATTCTGCCTGGTCCTNN SAAGNNSTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACANNSGTGNNSTTTGGCCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCTGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTGTAATGAGCTCAGAGAGGGTCCTGAT TTTCAGGGCCCTTTTTTTACGTGGTATTGTATAAAATGGATAAACCAAAGGCGTACTGCCGC GCAGTAGCGGTAAACGGCAGACAAAAAAAATGTCGCACAGTG SEQIDNO:41: CAGAATACGATCCAGTTTCCGSNNGTAGTTATACAGTGTCGG SEQIDNO:42: GGGTACAACCGCTACCCATCTGGCCAAASNNCACSNNTGTAAATTCTTCCAGGTGTT SEQIDNO:43:NucleicacidsequenceofCafRS#30 ATGGATAAAAAACCGCTGGATGTTCTGATTAGCGCAACAGGTCTGTGGATGAGCCGTACC GGCACCCTGCATAAAATCAAACATCACGAAGTTAGCCGCAGCAAAATCTATATTGAAATGG CCTGTGGCGATCATCTGGTTGTGAATAATAGCCGTAGCTGTCGTACCGCACGCGCATTTCG TCATCACAAATATCGTAAAACCTGTAAACGCTGCCGTGTGAGTGATGAAGATATTAACAATT TTCTGACACGCAGCACCGAAAGCAAAAATTCAGTTAAAGTTCGTGTTGTGAGCGCACCTAA AGTTAAAAAAGCAATGCCGAAAAGCGTTAGTCGCGCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGTAGCGTTCCGAGTCCGGCAAAAAGCACCCCG AATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCTTAACCCGGTCTCAGTTAGATCGT GTTGAAGCACTGCTGAGTCCTGAAGATAAAATCAGCCTGAATATGGCAAAACCGTTTCGTG AACTGGAACCGGAACTGGTTACTCGTCGTAAAAATGATTTTCAACGCTTATATACCAACGAC CGCGAAGATTATCTGGGTAAACTGGAACGTGATATTACCAAATTTTTCGTGGATCGCGGTT TTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGTATGGGCATTAA CAATGATACCGAACTGAGCAAACAAATCTTTCGCGTTGATAAAAACCTGTGTCTGCGTCCTA TGCTGACCCCGACATTGTATAACTACAGCCGGAAACTGGATCGTATTCTGCCTGGTCCTTC CAAAGTCTTTGAAGTTGGTCCGTGCTATCGTAAAGAAAGTGATGGTAAAGAACACCTGGAA GAATTTACAATGGTGGTGTTTGGCCAGATGGGTAGCGGTTGTACCCGTGAAAATCTGGAAG CACTGATTAAAGAGTTTCTGGATTACCTGGAAATCGACTTTGAAATTGTTGGTGATAGCTGC ATGGTTTTTGGTGATACCCTGGATATTATGCACGGTGATCTGGAACTGAGTAGCGCAGTTG TTGGTCCGGTTAGCCTGGATCGTGAATGGGGTATTGATAAACCCIGGATTGGTGCAGGTTT TGGTCTCGAACGTCTGCTGAAAGTAATGCACGGTTTCAAAAACATTAAACGTGCAAGCCGT TCCGAGAGCTACTATAATGGTATTAGCACCAACCTGTAA SEQIDNO:44:NucleicacidsequencepSAm1 GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAAT ATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAG TATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAG TACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACC GAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCA ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAAC AATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCA TTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTT TTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACG TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC GCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGC GGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCT TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACC GCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGATCTCGATCCCGCGAAATTAATAC GACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA GGAGATATACATATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACC TTCATCGTGACCGCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGGCGCGTGG CAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACG GCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCAC TCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCC AGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGC CACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCTAATAAGCTTGATCCGGCTGC TAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATA ACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCC GGATCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGC CTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTT CCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCAC GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC AAAAATTTAACGCGAATTTTAACAAATATTAACGCTTACAATTTAGGTG SEQIDNO:45: GCAGACGGaGCCCTGACCGGtACCTACTAGacggcgcgtG SEQIDNO:46: CacgcgccgtCTAGTAGGTaCCGGTCAGGGCtCCGTCTGC SEQIDNO:47:NucleicacidsequenceSAm1.sup.UAG44 ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC GCGGGTGCAGACGGAGCCCTGACCGGTACCTACTAGACGGCGCGTGGCAACGCCGAGA GCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACC GCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCAC GTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTG ACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTT CACCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:48: GATCAACACCCAGTAGCTGCTGACCTCC SEQIDNO:49: GGAGGTCAGCAGCTACTGGGTGTTGATC SEQIDNO:50: GAGGCCAACGCCTAGAAGTCCACGCTGG SEQIDNO:51: CCAGCGTGGACTTCTAGGCGTTGGCCTC SEQIDNO:52:NucleicacidsequenceSAm1.sup.UAG120 ATGGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACC GCGGGTGCAGACGGAGCTCTGACCGGTACCTACGTCACGGCGCGTGGCAACGCCGAGAG CCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCG CCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGT GGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGAC CTCCGGCACCACCGAGGCCAACGCCTAGAAGTCCACGCTGGTCGGCCACGACACCTTCA CCAAGGTGAAGCCGTCCGCCGCCTCCTAA SEQIDNO:53:NucleicacidsequenceofpSBX8.CafRS#30.d58 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGATAACGAGGGCAAAAAATGTCTAAAGGTGAAGAACTTTTCACTGGAGTT GTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGA GGGTGAAGGTGATGCAACATAGGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAA CTACCTGTTCCATGGCCAACACTTGTCACTACTTTGACTTATGGTGTTCAATGCTTTTCAAG ATACCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTA CAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGA AACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGA CAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCG TTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCA GACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGGGACC ACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTGTA CCAAAGCGCTTGGAGCCACCCGCAGTTCGAAAAATAATAAGCTTGACCTGTGAAGTGAAAA ATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGCGGCAGTACG CCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGGGCCCTGAAATTCAGGACCCTTT CTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAGCTCTCGGAACGGCTTGCACGT TTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTCGAGACCAAAACCTGCACCAAT CCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACCGGACCAACAACTGCGCTACTC AGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCAAAAACCATGCAGCTATCACCAA CAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTAATCAGTGCTTCCAGATTTTCA CGGGTACAACCGCTACCCATCTGGCCAAACACCACCATTGTAAATTCTTCCAGGTGTTCTT TACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAGACTTTGGAAGGACCAGGCAG AATACGATCCAGTTTCCGGCTGTAGTTATACAATGTCGGGGTCAGCATAGGACGCAGACAC AGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGTATCATTGTTAATGCCCATACG TTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTTCCAGAAAACCGCGATCCACG AAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCTTCGCGGTCGTTGGTATATAA GCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTTCCAGTTCACGAAACGGTTTT GCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCTTCAACACGATCTAACTGAGA CCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTGCTATTCGGGGTGCTTTTTGC CGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTGCGCTAACGCTATTTTCCAGA GGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTAACTTTAGGTGCGCTCACAA CACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAGAAAATTGTTAATATCTTCAT CACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGATGACGAAATGCGCGTGCGGT ACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCACAGGCCATTTCAATATAGATTT TGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTGCCGGTACGGCTCATCCACAG ACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCATATCGTACCTCCTTAAATTTC TAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGAAATGTCAAAAGAGAAGGGCG TGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGCGTCGGTGCCGATTTCGGCCT ATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGC AGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGATTTAGAGTCCATTCGATCTAC ATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAAGTTTTTTATGTTGAGAATATT TTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCATGCAGGTGGCACTTTTCGG GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTC CAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGG GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTT AATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAG CAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTG AGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTT TTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAG TACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTT TTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGGGCATTT TACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGG TGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTA AATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAGGAATAGAGGATATGGAG AAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGA GGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCC TTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCC CGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATA TGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCT CTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCG TGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTC AGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCT TCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCT GGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGCTTCACTAGTTTAAAAGG ATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC SEQIDNO:54:NucleicacidsequenceofpSBX8.CafRS#30.d47 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGATAACGAGGGCAAAAAATGGAAGCAGGTATCACCGGCACCTGGTACAA CCAGCTCGGCTCGACCTTCATCGTGACCGCGGGTGCAGACGGAGCCCTGACCGGTACCT ACTAGACGGCGCGTGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGC GCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAA CTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAG GCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAA GTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCTAATA AGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTA CCGCTACTGCGCGGCAGTACGCCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGG GCCCTGAAATTCAGGACCCTTTCTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAG CTCTCGGAACGGCTTGCACGTTTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTC GAGACCAAAACCTGCACCAATCCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACC GGACCAACAACTGCGCTACTCAGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCAA AAACCATGCAGCTATCACCAACAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTA ATCAGTGCTTCCAGATTTTCACGGGTACAACCGCTACCCATCTGGCCAAACACCACCATTG TAAATTCTTCCAGGTGTTCTTTACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAG ACTTTGGAAGGACCAGGCAGAATACGATCCAGTTTCCGGCTGTAGTTATACAATGTCGGGG TCAGCATAGGACGCAGACACAGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGT ATCATTGTTAATGCCCATACGTTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTT CCAGAAAACCGCGATCCACGAAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCT TCGCGGTCGTTGGTATATAAGCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTT CCAGTTCACGAAACGGTTTTGCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCT TCAACACGATCTAACTGAGACCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTG CTATTCGGGGTGCTTTTTGCCGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTG CGCTAACGCTATTTTCCAGAGGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTA ACTTTAGGTGCGCTCACAACACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAG AAAATTGTTAATATCTTCATCACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGAT GACGAAATGCGCGTGCGGTACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCAC AGGCCATTTCAATATAGATTTTGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTG CCGGTACGGCTCATCCACAGACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCA TATCGTACCTCCTTAAATTTCTAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGA AATGTCAAAAGAGAAGGGCGTGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGC GTCGGTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTT AGCTTTCGCTAAGGATCTGTCAGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGAT TTAGAGTCCATTCGATCTACATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAA GTTTTTTATGTTGAGAATATTTTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCA TGCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCITATTCCCTTTTTTGCGGCATTTTGC CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTT CCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT CACTGATTAAGCATTGGTAAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAAC AGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCC AGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCT CGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGG GAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCA TCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTC GAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTC AGCGCAGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTA AAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGA ATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCG GATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAG GAATAGAGGATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCA TCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTC AGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCC TTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAG ACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAAC TGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATAT ATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGG CCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGA CAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTC GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAG CTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCA GCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCC SEQIDNO:55:NucleicacidsequenceofpSBX8.CafRS#30.d53 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGATAACGAGGGCAAAAAATGGAAGCAGGTATCACCGGCACCTGGTACAA CCAGCTCGGCTCGACCTTCATCGTGACCGCGGGTGCAGACGGAGCTCTGACCGGTACCT ACGTCACGGCGCGTGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGC GCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAA CTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAG GCGAGGATCAACACCCAGTAGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAA GTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCTAATA AGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTA CCGCTACTGCGCGGCAGTACGCCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGG GCCCTGAAATTCAGGACCCTTTCTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAG CTCTCGGAACGGCTTGCACGTTTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTC GAGACCAAAACCTGCACCAATCCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACC GGACCAACAACTGCGCTACTCAGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCAA AAACCATGCAGCTATCACCAACAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTA ATCAGTGCTTCCAGATTTTCACGGGTACAACCGCTACCCATCTGGCCAAACACCACCATTG TAAATTCTTCCAGGTGTTCTTTACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAG ACTTTGGAAGGACCAGGCAGAATACGATCCAGTTTCCGGCTGTAGTTATACAATGTCGGGG TCAGCATAGGACGCAGACACAGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGT ATCATTGTTAATGCCCATACGTTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTT CCAGAAAACCGCGATCCACGAAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCT TCGCGGTCGTTGGTATATAAGCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTT CCAGTTCACGAAACGGTTTTGCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCT TCAACACGATCTAACTGAGACCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTG CTATTCGGGGTGCTTTTTGCCGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTG CGCTAACGCTATTTTCCAGAGGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTA ACTTTAGGTGCGCTCACAACACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAG AAAATTGTTAATATCTTCATCACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGAT GACGAAATGCGCGTGCGGTACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCAC AGGCCATTTCAATATAGATTTTGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTG CCGGTACGGCTCATCCACAGACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCA TATCGTACCTCCTTAAATTTCTAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGA AATGTCAAAAGAGAAGGGCGTGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGC GTCGGTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTT AGCTTTCGCTAAGGATCTGCAGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGAT TTAGAGTCCATTCGATCTACATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAA GTTTTTTATGTTGAGAATATTTTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCA TGCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGC CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTT CCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT CACTGATTAAGCATTGGTAAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAAC AGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCC AGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCT CGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGG GAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCA TCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTC GAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTC AGCGCAGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTA AAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGA ATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCG GATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAG GAATAGAGGATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCA TCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTC AGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCC TTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAG ACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAAC TGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATAT ATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGG CCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGA CAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTC GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAG CTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCA GCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCC SEQIDNO:56:NucleicacidsequenceofpSBX8.CafRS#30.d51 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGATAACGAGGGCAAAAAATGGAAGCAGGTATCACCGGCACCTGGTACAA CCAGCTCGGCTCGACCTTCATCGTGACCGCGGGTGCAGACGGAGCTCTGACCGGTACCT ACGTCACGGCGCGTGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGC GCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAA CTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAG GCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTAGAA GTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCTAATA AGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTA CCGCTACTGCGCGGCAGTACGCCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGG GCCCTGAAATTCAGGACCCTTTCTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAG CTCTCGGAACGGCTTGCACGTTTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTC GAGACCAAAACCTGCACCAATCCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACC GGACCAACAACTGCGCTACTCAGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCAA AAACCATGCAGCTATCACCAACAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTA ATCAGTGCTTCCAGATTTTCACGGGTACAACCGCTACCCATCTGGCCAAACACCACCATTG TAAATTCTTCCAGGTGTTCTTTACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAG ACTTTGGAAGGACCAGGCAGAATACGATCCAGTTTCCGGCTGTAGTTATACAATGTCGGGG TCAGCATAGGACGCAGACACAGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGT ATCATTGTTAATGCCCATACGTTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTT CCAGAAAACCGCGATCCACGAAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCT TCGCGGTCGTTGGTATATAAGCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTT CCAGTTCACGAAACGGTTTTGCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCT TCAACACGATCTAACTGAGACCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTG CTATTCGGGGTGCTTTTTGCCGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTG CGCTAACGCTATTTTCCAGAGGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTA ACTTTAGGTGCGCTCACAACACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAG AAAATTGTTAATATCTTCATCACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGAT GACGAAATGCGCGTGCGGTACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCAC AGGCCATTTCAATATAGATTTTGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTG CCGGTACGGCTCATCCACAGACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCA TATCGTACCTCCTTAAATTTCTAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGA AATGTCAAAAGAGAAGGGCGTGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGC GTCGGTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTT AGCTTTCGCTAAGGATCTGCAGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGAT TTAGAGTCCATTCGATCTACATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAA GTTTTTTATGTTGAGAATATTTTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCA TGCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGC CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTT CCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT CACTGATTAAGCATTGGTAAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAAC AGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCC AGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCT CGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGG GAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCA TCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTC GAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTC AGCGCAGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTA AAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGA ATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCG GATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAG GAATAGAGGATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCA TCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTC AGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCC TTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAG ACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAAC TGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATAT ATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGG CCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGA CAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTC GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAG CTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCA GCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCC SEQIDNO:57:NucleicacidsequenceofpASK75-PhoA-strepII CCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTAT TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGAAC ATGGAGAAAATAAAGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACC CCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGC GATATTACTGCACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGT GATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGG ACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTAT AGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAAAAAAACCGGCAAA CCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACC TATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATG GCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACG CCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAG TGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCT GCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGC AACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATC AGTTGGTGAGCGATGCTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCAAAAACCCC TGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGT ACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAG TGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAA GGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTT GTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAATTC GCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAG ATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGC GCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGT CAGTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAG ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACCGCCTAGCGCTTGGTCT CACCCGCAGTTCGAAAAATAATAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGC GACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGTCACGGATCTCCACGCGCCCTGTAGC GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAG CGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCTTTCTCGCCACGTTCGCCGGCTTT CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGAC GGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTG GAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCG GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTA ACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATT TTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA ATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG CGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATT CTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGAC AGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCAT GTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT GACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA GCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA GATAGGTGCCTCACTGATTAAGCATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAGTA AAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCG TAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAG CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCC CTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCT TTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGT ATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCAGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGC ATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACG ACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAAT TGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCATAAC CTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAA AAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC GCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGA AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT GACCCGACA SEQIDNO:58:Nucleicacidsequenceofhumanalbuminbindingdomain(ABD) CTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTGAGCGACTATT ACAAGAACCTGATTAATAACGCGAAAACCGTGGAAGGTGTTAAAGCACTGATTGATGAAAT TCTGGCAGCACTGCCG SEQIDNO:59:Aminoacidsequenceofhumanalbuminbindingdomain(ABD) LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP SEQIDNO:60:NucleicacidsequenceofProtL-ABDfusionprotein ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCTATGCCGATCTGCTG GCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCA AATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:61:AminoacidsequenceofProtL-ABDfusionprotein. Methionine(underlined)wasaddedasastartcodon. MKEEVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFAG GAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP

[0295] In the following, for illustration purposes, the amino acid sequence of ProtL-ABD (SEQ ID NO: 61) is shown below the corresponding nucleic acid sequence (SEQ ID NO: 60). The sequence of protein L domain B1 begins with Lys.sup.326 and ends with Gly.sup.389 (UniProt Q51918)

[0296] The position of 337, 347, 360, 364, 368 and 369 are in bold face and underlined.

TABLE-US-00010 102030405060 ++++++ 1ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACC60 MetLysGluGluValThrIleLysValAsnLeuIlePheAlaAspGlyLysThrG1nThr 326 708090100110120 ++++++ 61GCAGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCTATGCCGATCTG120 AlaGluPheLysGlyThrPheGluGluAlaThrAlaLysAlaTyrAlaTyrAlaAspLeu 130140150160170180 ++++++ 121CTGGCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAAT180 LeuAlaLysGluAsnGlyGluTyrThrAlaAspLeuGluAspGlyGlyAsnThrIleAsn 190200210220230240 ++++++ 181ATCAAATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCA240 IleLysPheAlaGlyGlyAlaValAspAlaAsnSerLeuAlaGluAlaLysValLeuAla 389>albuminbindingdomain 250260270280290300 ++++++ 241AATCGTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCG300 AsnArgGluLeuAspLysTyrGlyValSerAspTyrTyrLysAsnLeuIleAsnAsnAla 310320330340350 +++++ 301AAAACCGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAG354 LysThrValGluGlyValLysAlaLeuIleAspGluIleLeuAlaAlaLeuPro SEQIDNO:62:NucleicacidsequenceofpASK75-ProtL-ABD CCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTAT TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAATGAATAGTTCGACAAAAATCTAGAAA TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAGAAGAAGTTACCATTAAAGTTAA TCTGATTTTCGCCGATGGTAAAACCCAGACCGCAGAATTTAAAGGCACCTTTGAAGAAGCA ACCGCAAAAGCCTATGCCTATGCCGATCTGCTGGCAAAAGAAAATGGTGAATATACCGCAG ATCTGGAAGATGGTGGTAATACCATCAATATCAAATTTGCCGGTGGTGCCGTTGATGCAAA TAGCCTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTGAGCGAC TATTACAAGAACCTGATTAATAACGCGAAAACCGTGGAAGGTGTTAAAGCACTGATTGATG AAATTCTGGCAGCACTGCCGTAATAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGT GCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGTCACGGATCTCCACGCGCCCTGTA GCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCT TTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAA CTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATA TTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCA ATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTT TGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATG TGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTA TTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG ACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTAC TTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGC GTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT ACTTACTCTAGCTTCCCGGCAACAATTgATAGACTGGATGGAGGCGGATAAAGTTGCAGGA CCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGT GAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT GAGATAGGTGCCTCACTGATTAAGCATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAG TAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACC CGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATA AGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTG CCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTG CTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACA GTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATG CATTATATGCACTCAGCGCaGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGA GCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTA CGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTG AATTGATCATtTGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCAT AACCTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGA TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGT AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT CACATGACCCGACA SEQIDNO:63:NucleicacidsequenceofAJR_ProtL_PHE337TAG_fw CATTAAAGTTAATCTGATTTAGGCCGATGGTAAAAC SEQIDNO:64:NucleicacidsequenceofAJR_ProtL_PHE337TAG_rv GTTTTACCATCGGCCTAAATCAGATTAACTTTAATG SEQIDNO:65:NucleicacidsequenceofAJR_ProtL_Y361N_L365S_fw CAAAAGCCTATGCCAACGCCGATCTGAGCGCGAAAATGGTG SEQIDNO:66:NucleicacidsequenceofAJR_ProtL_Y361N_L365S_rv CACCATTTTCTTTTGCGCTCAGATCGGCGTTGGCATAGGCTTTTG SEQIDNO:67:NucleicacidsequenceofProtL.sup.UAG337-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTAGGCCGATGGTAAAACCCAGACCG CAGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCAACGCCGATCTGAG CGCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATC AAATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:68:NucleicacidsequenceofAJR_ProtL_PHE347TAG_fw CAGACCGCAGAATAGAAAGGCACCTTTG SEQIDNO:69:NucleicacidsequenceofAJR_ProtL_PHE347TAG_rv CAAAGGTGCCTTTCTATTCTGCGGTCTG SEQIDNO:70:NucleicacidsequenceofAJR_ProtL_TYR361ALA_fw CCGCAAAAGCCTATGCCGCGGCCGATCTGCTGGC SEQIDNO:71:NucleicacidsequenceofAJR_ProtL_TYR361ALA_rv GCCAGCAGATCGGCCGCGGCATAGGCTTTTGCGG SEQIDNO:72:NucleicacidsequenceofProtL.sup.UAG347-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATAGAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCGCGGCCGATCTGCT GGCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATC AAATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAATC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:73:NucleicacidsequenceofAJR_ProtL_ALA360TAG_fw CCGCAAAAGCCTATTAGTATGCCGATCTGC SEQIDNO:74:NucleicacidsequenceofAJR_ProtL_ALA360TAG_rv GCAGATCGGCATACTAATAGGCTTTTGCGG SEQIDNO:75:NucleicacidsequenceofProtL.sup.UAG360-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATTAGTATGCCGATCTGCTG GCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCA AATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:76:NucleicacidsequenceofAJR_ProtL_LEU364TAG_fw CTATGCCTATGCCGATTAGCTGGCAAAAGAAAATGG SEQIDNO:77:NucleicacidsequenceofAJR_ProtL_LEU364TAG_rv CCATTTTCTTTTGCCAGCTAATCGGCATAGGCATAG SEQIDNO:78:NucleicacidsequenceofProtL.sup.UAG364-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCTATGCCGATTAGCTG GCAAAAGAAAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCA AATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:79:NucleicacidsequenceofAJR_ProtL_GLU368TAG_fw GATCTGCTGGCAAAATAGAATGGTGAATATACCG SEQIDNO:80:NucleicacidsequenceofAJR_ProtL_GLU368TAG_rv CGGTATATTCACCATTCTATTTTGCCAGCAGATC SEQIDNO:81:NucleicacidsequenceofProtL.sup.UAG368-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCTATGCCGATCTGCTG GCAAAATAGAATGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCA AATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:82:NucleicacidsequenceofAJR_ProtL_ASN369TAG_fw CTGCTGGCAAAAGAATAGGGTGAATATACCGC SEQIDNO:83:NucleicacidsequenceofAJR_ProtL_ASN369TAG_rv GCGGTATATTCACCCTATTCTTTTGCCAGCAG SEQIDNO:84:NucleicacidsequenceofProtL.sup.UAG369-ABD ATGAAAGAAGAAGTTACCATTAAAGTTAATCTGATTTTCGCCGATGGTAAAACCCAGACCGC AGAATTTAAAGGCACCTTTGAAGAAGCAACCGCAAAAGCCTATGCCTATGCCGATCTGCTG GCAAAAGAATAGGGTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCA AATTTGCCGGTGGTGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATC GTGAACTGGATAAATATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAAC CGTGGAAGGTGTTAAAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAA SEQIDNO:85:NucleicacidsequenceofpSBX8.CafRS#30d71 TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATAACGCTC GGTTGCCGCCGGGCGTTTTTTATTGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTA TCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTC GACAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAGAAGAAGT TACCATTAAAGTTAATCTGATTTAGGCCGATGGTAAAACCCAGACCGCAGAATTTAAAGGCA CCTTTGAAGAAGCAACCGCAAAAGCCTATGCCAACGCCGATCTGAGCGCAAAAGAAAATG GTGAATATACCGCAGATCTGGAAGATGGTGGTAATACCATCAATATCAAATTTGCCGGTGG TGCCGTTGATGCAAATAGCCTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAA TATGGTGTGAGCGACTATTACAAGAACCTGATTAATAACGCGAAAACCGTGGAAGGTGTTA AAGCACTGATTGATGAAATTCTGGCAGCACTGCCGTAATAAGCTTGACCTGTGAAGTGAAA AATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGCGGCAGTAC GCCTTTGGTTTATCCATTTTATACAATCCATGTAAAAAAGGGCCCTGAAATTCAGGACCCTT TCTGAGCTCATTACAGGTTGGTGCTAATACCATTATAGTAGCTCTCGGAACGGCTTGCACG TTTAATGTTTTTGAAACCGTGCATTACTTTCAGCAGACGTTCGAGACCAAAACCTGCACCAA TCCAGGGTTTATCAATACCCCATTCACGATCCAGGCTAACCGGACCAACAACTGCGCTACT CAGTTCCAGATCACCGTGCATAATATCCAGGGTATCACCAAAAACCATGCAGCTATCACCA ACAATTTCAAAGTCGATTTCCAGGTAATCCAGAAACTCTTTAATCAGTGCTTCCAGATTTTCA CGGGTACAACCGCTACCCATCTGGCCAAACACCACCATTGTAAATTCTTCCAGGTGTTCTT TACCATCACTTTCTTTACGATAGCACGGACCAACTTCAAAGACTTTGGAAGGACCAGGCAG AATACGATCCAGTTTCCGGCTGTAGTTATACAATGTCGGGGTCAGCATAGGACGCAGACAC AGGTTTTTATCAACGCGAAAGATTTGTTTGCTCAGTTCGGTATCATTGTTAATGCCCATACG TTCAACATATTCTGCCGGAATCAGAATCGGGCTTTTGATTTCCAGAAAACCGCGATCCACG AAAAATTTGGTAATATCACGTTCCAGTTTACCCAGATAATCTTCGCGGTCGTTGGTATATAA GCGTTGAAAATCATTTTTACGACGAGTAACCAGTTCCGGTTCCAGTTCACGAAACGGTTTT GCCATATTCAGGCTGATTTTATCTTCAGGACTCAGCAGTGCTTCAACACGATCTAACTGAGA CCGGGTTAAGCTCGGTGCCGGTGCGCTTGCCGGAACGCTGCTATTCGGGGTGCTTTTTGC CGGACTCGGAACGCTACGGCTGGTATTGGTGCTTGCTTTTGCGCTAACGCTATTTTCCAGA GGTTTCGGTGCGCGACTAACGCTTTTCGGCATTGCTTTTTTAACTTTAGGTGCGCTCACAA CACGAACTTTAACTGAATTTTTGCTTTCGGTGCTGCGTGTCAGAAAATTGTTAATATCTTCAT CACTCACACGGCAGCGTTTACAGGTTTTACGATATTTGTGATGACGAAATGCGCGTGCGGT ACGACAGCTACGGCTATTATTCACAACCAGATGATCGCCACAGGCCATTTCAATATAGATTT TGCTGCGGCTAACTTCGTGATGTTTGATTTTATGCAGGGTGCCGGTACGGCTCATCCACAG ACCTGTTGCGCTAATCAGAACATCCAGCGGTTTTTTATCCATATCGTACCTCCTTAAATTTC TAGGTTGTGACCTAGGTGATTTAGTTTACCAGTGCAAAAGAAATGTCAAAAGAGAAGGGCG TGAATTTAACGCGGTTCCAGCGCAAAGACTTCAAAACCTGCGTCGGTGCCGATTTCGGCCT ATTGGTTAAAAAATGAGCTGAGTTCTAGTAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGC AGTGGCGGAAACCCCGGGAATCTAACCCGGCTGAACGGATTTAGAGTCCATTCGATCTAC ATGATCAGGTTTCCGAATTCAGCGTTACAAGTATTACACAAAGTTTTTTATGTTGAGAATATT TTTTTGATGGGGCATGGCGCAAAACCTTTCGCGGTATGGCATGCAGGTGGCACTTTTCGG GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTC CAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGG GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AAGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTT AATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAG CAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTG AGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTT TTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAG TACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTT TTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGGGCATTT TACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGG TGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTA AATGTGAAAGTGGGTCTTAATGAGAATATTCGTTTTCACCCAAGGAATAGAGGATATGGAG AAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGA GGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCC TTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCC CGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATA TGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCT CTGGAGTGAATACCACGACTAGTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCG TGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTC AGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCT TCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCT GGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGCTTCACTAGTTTAAAAGG ATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAACGCCAGCAACGCGGCC SEQIDNO:86:aminoacidsequenceofProtL.sup.UAG337-ABD. ThepositionofCafisinboldfaceandunderlined. MetLysGluGluValThrIleLysValAsnLeuIleCafAlaAspGlyLysThrGlnThr AlaGluPheLysGlyThrPheGluGluAlaThrAlaLysAlaTyrAlaAsnAlaAspLeu SerAlaLysGluAsnGlyGluTyrThrAlaAspLeuGluAspGlyGlyAsnThrIleAsn IleLysPheAlaGlyGlyAlaValAspAlaAsnSerLeuAlaGluAlaLysValLeuAla AsnArgGluLeuAspLysTyrGlyValSerAspTyrTyrLysAsnLeuIleAsnAsnAla LysThrValGluGlyValLysAlaLeuIleAspGluIleLeuAlaAlaLeuPro