THERMOSTABLE GLUCOSE BIOSENSORS AND USES THEREOF
20230194538 · 2023-06-22
Inventors
Cpc classification
G01N21/6428
PHYSICS
G01N2800/042
PHYSICS
G01N33/542
PHYSICS
International classification
Abstract
The present subject matter provides glucose biosensors as well as compositions, devices, and methods comprising such biosensors.
Claims
1. A biosensor for glucose, comprising a glucose-binding protein and a reporter group that transduces a detectable signal, wherein the reporter group is attached to the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose, wherein the glucose-binding protein does not comprise a Ca.sup.2+ binding site, and wherein the glucose-binding protein does not comprise an enzyme.
2. The biosensor of claim 1, wherein the glucose-binding protein comprises amino acids in the sequence set forth as SEQ ID NO: 48 or 56, and wherein Acrylodan is attached to a cysteine of said glucose-binding protein.
3. (canceled)
4. The biosensor of claim 1, wherein the glucose-binding protein comprises a mutation compared to a naturally occurring protein, wherein at least one amino acid of the naturally occurring protein has been substituted with a cysteine.
5. The biosensor of claim 1, wherein the glucose-binding protein comprises a mutation compared to a naturally occurring protein, wherein the glucose-binding protein has no deletions or insertions compared to the naturally occurring protein.
6. The biosensor of claim 1, wherein the glucose-binding protein comprises a mutation compared to a naturally occurring protein, wherein the glucose-binding protein comprises (i) less than about 5, 4, 3, 2, or 1 inserted amino acids, and/or (ii) less than about 5, 4, 3, 2, or 1 deleted amino acids compared to the naturally occurring protein.
7. The biosensor of claim 1, wherein the glucose-binding protein comprises a mutant of a microbial glucose-binding protein.
8. The biosensor of claim 7, wherein the mutant comprises a mutation that alters the mutant's affinity and/or specificity for glucose compared to the microbial glucose-binding protein.
9. The biosensor of claim 1, wherein the amino acid sequence of said glucose-binding protein is less than 20% identical to the amino acid sequence of E. coli glucose-galactose binding protein (ecGGBP; SEQ ID NO: 117).
10. (canceled)
11. (canceled)
12. The biosensor of claim 1, wherein the glucose-binding protein comprises a mutation compared to a naturally occurring protein, wherein the naturally occurring protein is from an archaean microorganism, a Gram-positive bacterium, or a Gram-negative bacterium.
13. The biosensor of claim 1, wherein the glucose-binding protein comprises or comprises a mutant of: a Thermus sp. glucose-binding protein, a Thermotoga sp. glucose-binding protein, a Kosmotoga sp. glucose-binding protein, or a Staphylothermus sp. glucose-binding protein.
14. The biosensor of claim 1, wherein the glucose-binding protein comprises or comprises a mutant of: a Deinococcus sp. glucose-binding protein, a Bacillus sp. glucose-binding protein, or a Arthrobacter sp. glucose-binding protein.
15. The biosensor of claim 1, wherein the glucose-binding protein comprises or comprises a mutant of: a glucose-binding protein from Thermus thermophilus (ttGBP1; SEQ ID NO: 1, 9, or 109); a glucose-binding protein from Thermus scotoductus (tsGBP2; SEQ ID NO: 2, 10, or 110); a glucose-binding protein from Deinococcus maricopensis (dmGBP3; SEQ ID NO: 3, 11, or 111); a glucose-binding protein from Thermotoga neapolitana (tnGBP4; SEQ ID NO: 4, 12, or 112); a glucose-binding protein from Kosmotoga olearia (koGBP5; SEQ ID NO: 5, 13, or 113); a glucose-binding protein from Bacillus halodurans (bhGBP6; SEQ ID NO: 6, 14, or 114); a glucose-binding protein from Staphylothermus marinus (smGBP7; SEQ ID NO: 7, 15, or 115); or a glucose-binding protein from Arthrobacter sp. (asGBP8; SEQ ID NO: 8, 16, or 116).
16. The biosensor of claim 1, wherein the glucose-binding protein comprises an amino acid sequence that is between 10% and 100% identical to the amino acid sequence of ttGBP1, tsGBP2, dmGBP3, tnGBP4, koGBP5, bhGBP6, smGBP7, or asGBP8.
17.-22. (canceled)
23. The biosensor of claim 1, wherein the Ca root-mean-square deviation (RMSD) between the backbone of the glucose-binding polypeptide and ttGBP1, tsGBP2, dmGBP3, tnGBP4, koGBP5, bhGBP6, smGBP7, or asGBP8 is between about 0-3 Å, 0-1 Å, 0-1.5 Å, 0-2 Å, 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å.
24. (canceled)
25. The biosensor of claim 1, wherein the glucose-binding protein is a mutant of tsGBP2 comprising one or more of the following substitutions: W8X, W9X, D12X, E13X, G41X, A42X, Q64X, H66X, H119X, W167X, 5223X, W224X, Q225X, W244X, 5277X, D278X, K312X, W337X, H348X, and M357C, wherein X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in tsGBP2 with the signal peptide replaced with a methionine (SEQ ID NO: 10 or 110).
26.-31. (canceled)
32. The biosensor of claim 1, wherein the reporter group is covalently attached to the glucose-binding protein.
33. (canceled)
34. (canceled)
35. (canceled)
36. The biosensor of claim 1, wherein the reporter group is conjugated to a cysteine of the glucose-binding protein.
37. (canceled)
38. The biosensor of claim 1, wherein the reporter group comprises a fluorophore.
39.-52. (canceled)
53. A method of detecting the presence or concentration of glucose in a sample, the method comprising: (a) contacting the biosensor of claim 1 with the sample; (b) measuring a signal from the biosensor; and (c) comparing the signal to a glucose control value, wherein a difference in signal indicates the presence of glucose in the sample.
54.-74. (canceled)
75. A method for monitoring the level of glucose in a subject, comprising (a) administering a biosensor according to claim 1 or a device comprising a biosensor according to claim 1 to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface of the subject, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart.
76.-122. (canceled)
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0379] Microbes have separately evolved different types of proteins that bind to glucose in what can be seen as an example of convergent evolution. Across these types of proteins, glucose-binding involves a large hinge-bending motion that transitions the proteins from an open to a closed state in which the glucose is enveloped within a cleft between two domains. Multiple structural classes of bacterial proteins that bind glucose have been categorized based on the ordering of β-strands within each domain (
[0380] Fluorescently responsive sensors (FRSs) based on engineered (i.e., produced by artificial selection, design, mutation, conjugation, and/or other human-directed activity) proteins that couple ligand-binding events to changes in the emission properties of fluorophores (being fluorescent by themselves and regardless of the presence of any other fluorophore/partner) or semi-synthetically incorporated chromophores have wide-ranging applications in cell biology and analytical chemistry. If the fluorescence emission spectrum of an engineered FRS changes shape in response to ligand binding such that the ratio of intensities at two appropriately chosen wavelengths reports on ligand concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations (
[0381] The periplasmic binding protein (PBP) superfamily provide a rich source of FRSs, because PBPs combine a large diversity of ligand specificities with a common structural mechanism that is well suited to the construction of fluorescence signal transduction schemes. The three-dimensional PBP monomer structure comprises two α/β domains linked by a β-strand hinge. Different PBP structural classes have been categorized based on the ordering of β-strands within each domain (
[0382] A glucose-binding protein has been identified in the hyperthermophilic bacterium Thermus thermophilus (ttGBP1) (Class II). This protein is homologous to a group of periplasmic-binding proteins that are adaptations of the E. coli maltose-binding protein and are structurally quite distinct from the ecGGBP proteins (
[0383] Significance of a given alignment is an important question in constructing sets of sequence homologs has been addressed in the art (e.g., in D. W. Mount, 2001, “Bioinformatics”, Cold Spring Harbor Laboratory Press, the entire content of which is incorporated herein by reference). One approach is to assess whether the alignment score of a particular sequence pair is significantly different from a random pair of sequences with the same amino composition and gap distribution. The BLAST program generates a list of possible pairs of aligned sequence fragments whose score cannot be improved upon by extending or trimming. For each such “high-scoring segment”, HSP, its expectation value, E, that the match is random is reported. For values ≤0.01, the E value corresponds to the classical P value, the probability of the null hypothesis (i.e. probability of a random match). Small values of E (0.01 or less) correspond to significant matches: the closer to 0, the more significant the match (i.e. the probability that the match is random is close to 0).
[0384] In the case of aligning ecGGBP (Genbank Accession No. YP_003350022.1) with ttGPB1 (NCBI Accession No. YP_004303.1) the HSP has an E value greater than 1.6. In other words, the alignment of these two sequences shows that they are about as similar as two random and unrelated sequences, e.g., the alignments of these two sequences are indistinguishable from a random alignment.
[0385] Here we present the construction of semisynthetic, reagentless, ratiometric fluorescent glucose biosensors based on the hyperthermophilic glucose-binding protein homolog of ttGBP1 identified in Thermus scotoductus (tsGBP2). These engineered tsGBP conjugates respond to glucose concentrations in clinically relevant concentration ranges (from ˜1 mM in extreme hypoglycemia, to ˜100 mM for the hyperosmolar, hyperglycemic condition, with healthy, euglycemic levels at ˜6 mM). The thermostability of these proteins exceeds 100° C. Furthermore, the selectivity of tsGBP2 has been engineered such that a sensor with a glucose affinity of about 5.6 mM, which is near-optimal for sensing in the euglycemic concentration range, has an affinity for galactose of about 140 mM (see, e.g.,
[0386] Glucose monitoring is essential for the management of diabetes mellitus, a disease that affects at least 366 million people world-wide and is increasing every year. The majority of current glucose-monitoring technologies rely on enzymes for which glucose is one of the substrates. Glucose concentration measurements are therefore subject to variations in second substrate concentrations consumed in the enzyme reaction, such as oxygen in the case of glucose oxidase. Additional complications arise in systems where reaction rates are measured for enzymes immobilized on electrodes. In such arrangements, accuracy is compromised by factors that alter the rate at which glucose arrives at the electrode surface interfere with accuracy, such as hematocrit levels, or surface “fouling” by deposition of proteins and cells in the foreign body response. Ratiometric fluorescent glucose sensors obviate these problems, and accordingly have been incorporated successfully in optodes for continuous glucose monitoring in animals and humans.
[0387] In FRS-based sensors, signals arise from reversible binding equilibria of the analyte (ligand) to a receptor. These signals are most precise at ligand concentrations that match the receptor ligand-disassociation constant. Precision is maintained to within ˜80% of this maximal level over a concentration range approximately 3-fold above or below this point. Construction of effective FRS therefore requires matching of ligand-binding affinities to the relevant analyte concentrations. Arrays of multiple sensors may have to be used in concert to cover wide concentration ranges. Clinically relevant glucose levels vary approximately 100-fold (from ˜1 mM in extreme hypoglycemia, to ˜100 mM for the hyperosmolar, hyperglycemic condition, with healthy, euglycemic levels at ˜6 mM (American Diabetes Association 2000 Clinical Diabetes, 18; Pasquel 2014 Diabetes Care, 37, 3124-3131), requiring an array of multiple FRS sensors with distinct glucose affinities to report directly on the full range of clinically relevant glucose concentrations with high precision. Here we report a set of appropriately tuned hyperthermostable, glucose-responsive FRSs, constructed by mutating their glucose-binding site.
[0388] Immobilization of FRSs on solid surfaces with minimal perturbation of the molecular sensing mechanism is an important step for incorporating biosensors into devices. Immobilization enables retention of the sensor within the sampling element (e.g. optode surface or implanted bead for in vivo sensing applications; or in a sample-handling cartridge for ex vivo sensing). Immobilization also may provide spatial localization to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of glucose concentrations, or sensors that each detect distinct analytes.
[0389] Ex vivo clinical chemistries such as point-of-care applications require that the FRS is incorporated into a cartridge into which a sample is introduced at the time of measurement. Such “disposables” need to have a long shelf life that preferably does not require temperature control (e.g. refrigeration) for storage or distribution. It is preferable to incorporate immobilized protein in a stable, dried form in such disposables. The inherent resistance to denaturation of thermostable proteins minimizes the need for temperature control during manufacturing and storage, and may extend to the long-term stability of a desiccated state.
[0390] The spectral response and thermostability of the robust thermostable glucose FRSs reported here are conserved following site-specific immobilization on beads or other solid substrates. Furthermore, these properties are recovered rapidly upon reconstitution following drying and prolonged storage under accelerated aging conditions. These engineered proteins are therefore useful for the development of robust, high-precision, wide-dynamic range glucose sensing applications, including continuous monitoring, point-of-care, wearable sensor systems.
Biosensors
[0391] Biosensors are molecular recognition elements that transduce ligand-binding events into physical detectable signals. Biosensors as detailed herein bind at least one ligand and emit a detectable signal such as fluorescence. A ligand-bound biosensor results in a signal that is different from a signal from the corresponding unbound biosensor. This difference facilitates detection of the at least one ligand and/or determination of ligand concentration. The biosensors may be used without the presence or assistance of other reagents.
[0392] The present subject matter provides improved biosensors that rapidly, reliably, and accurately detect and quantify glucose with significant advantages over previous systems. Aspects include a biosensor for glucose, comprising a reporter group that is attached to a glucose-binding protein. The glucose comprises glucose, and the glucose-binding protein includes a domain or region(s) that binds the glucose. The domain or region involved in ligand binding is comprised of a plurality of residues, e.g., non-contiguous amino acids of the ligand-binding protein, which are contact points or sites of contact between the ligand and its cognate ligand-binding protein. The binding of a glucose to the glucose-binding domain of the glucose-binding protein causes a change in signaling by the reporter group. In various implementations, the biosensor may produce a signal when a glucose is bound to the glucose binding domain that is not produced (and/or that is different from a signal that is produced) when the glucose is absent from the glucose binding domain. These biosensors have widespread utility including in clinical, food and beverage, industrial, and environmental settings.
[0393] A reporter group that transduces or emits a detectable signal may be attached to the glucose-binding proteins (biosensors) described herein. As used herein, “transduce” means the conversion of ligand occupancy in the binding site of a ligand-binding protein to a detectable signal. Occupancy refers to the state of ligand being bound or not bound to a cognate ligand-binding protein. In embodiments, detectable signal comprises a fluorescent, electrochemical, nuclear magnetic resonance (NMR), or electron paramagnetic resonance (EPR) signal. The reporter group is attached to the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose. The proteins may be engineered to include a single cysteine to which the detectable label, e.g., a fluorophore is covalently attached. The biosensors are reagentless in that their monitoring mechanism requires neither additional substrates for a signal to develop, nor measurement of substrate consumption or product generation rates to determine glucose concentrations.
[0394] Binding of ligand mediates conformational changes in the biosensor, such as hinge-bending motions of the polypeptide. The conformational changes affect the environment of the reporter such that a change in the reporter-generated signal occurs. That is, without ligand bound, the biosensor results in signal generated from the reporter, and when ligand is bound, the signal generated from the reporter changes. The ligand-bound biosensor results in a reporter-generated signal that is different from the unbound biosensor. For example, the spectral shape of the tsGBP2 13C⋅Acrylodan W244F biosensor changes when the biosensor becomes bound to glucose (see
[0395] In some embodiments, the methods and compositions include a plurality of a single type of biosensor. The biosensors may be identical in structure and function. For example, the biosensors of a single type may have the same polypeptide, the same reporter, and the same ligand affinity.
[0396] In other embodiments, the methods and compositions include a plurality of different types of bio sensors. A plurality of these different types of biosensors may be arranged or incorporated in a panel. As used herein, a “panel” refers to two or more biosensors. The two or more biosensors may be different from each other. The biosensors may differ in structure and/or function. Biosensors may differ in polypeptide sequence, reporter, ligand affinities, or a combination thereof. Accordingly, there may be different types of biosensors. In some embodiments, each biosensor in the panel comprises the same reporter group. In some embodiments, each biosensor in the panel comprises a different reporter group. The panel may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 biosensors.
[0397] The panel of biosensors includes at least one sensor element. “Sensor element” refers to a single spot, site, location, or well for the at least one biosensor, to which a sample or aliquot thereof may be applied. The panel may be a composite sensor or an array.
[0398] In some embodiments, the panel is a composite sensor. In a composite sensor, each sensor element includes a mixture of two or more different biosensors. In some embodiments, the composite sensor includes one sensor element. In some embodiments, the composite sensor includes two or more sensor elements. In some embodiments, signals are measured from a composite sensor in which the signals arise from one or more biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from a subset of the total number of biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from two of five biosensors in the sensor element.
[0399] In some embodiments, the panel is an array. In an array, each sensor element includes a single type of biosensor. An array comprises a plurality of individually and spatially localized sensor elements. Each sensor element includes a biosensor that is different than or the same as the bio sensor of a different sensor element. In some embodiments, signals are measured from an array in which the signals arise separately from two or more selected biosensors in separate sensor elements. An array may comprise a plurality of sensor elements of a variety of sizes and configurations. An array may comprise a plurality of sensor elements arranged linearly. For example, an array may comprise a plurality of micrometer-sized sensor elements arranged in a single row. An array may comprise a plurality of sensor elements arranged in a grid. The grid may be two- or three-dimensional. In some embodiments, the grid is a spatially addressable grid. In some embodiments, the biosensors are incorporated into an array, such as a multichannel or multiplexed array.
[0400] The biosensors of the present disclosure can be used in any setting where glucose detection is required or desired, such a medical setting (e.g., determining the level of blood glucose in a subject), environmental setting (e.g., determining the level of glucose in an environmental sample), biological setting (e.g., determining the presence or amount of glucose in a reaction), or in process engineering, such as monitoring the amount of glucose in a fermentation reaction (e.g., a bacterial culture, a yeast culture, beer/wine production, etc.). Other examples include, but are not limited to, uses in the food industry (Suleiman et al, In: Biosensor Design and Application: Mathewson and Finley Eds; American Chemical Society, Washington, D.C. 1992, vol. 511); in clinical chemistry (Wilkins et al., Med. Eng. Phys. 1996, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al., Endricon 1966, 6, 51-58; Riklin et al., Nature 1995, 376, 672-675); Willner et al., J. Am. Chem. Soc. 1996, 118, 10321-10322); as the basis for the construction of a fluorescent flow cell containing immobilized ligand binding protein-FAST conjugates (see, e.g., Wilkins et al., Med. Eng. Phys. 1966, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al., Endricon. 1966, 6, 51; Group, New Engl. J. Med. 1993, 329, 977-986; Gough et al., Diabetes 1995, 44, 1005-1009); and in an implantable devices.
[0401] The biosensors as detailed herein may be administered in a variety of ways known by those of skill in the art, as appropriate for each application. Biosensors may be provided in a solution. The solution may be buffered. Biosensors may be provided in a solution and mixed directly with a sample. In some embodiments, a biosensor is immobilized onto a surface. Biosensors may be immobilized within a disposable cartridge into which a sample may be introduced or applied. Biosensors may be implanted or incorporated in a wearable device. The biosensor may be provided as an optode.
[0402] The biosensor may be attached to or incorporated in a wearable device. Wearable devices may include, for example, adhesive strips, patches, and contact lenses. The biosensor may be configured for placement in contact with a subject's skin or mucosal surface. In some embodiments, the biosensor is configured as an adhesive strip. In some embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the contact lens is formed from a transparent substrate shaped to be worn directly over a subject's eye, as described in, for example, U.S. Pat. No. 8,608,310.
[0403] The biosensor may be implanted. The biosensor may be implanted in a subject's body. The biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, skin, or anywhere in the alimentary canal including the stomach, intestine and esophagus. The biosensor may be implanted in a subject with a microbead. In some embodiments, the biosensor is configured to be implanted in the skin. The biosensor may be implanted in a subject sub-dermally. The biosensor may generate the signal trans-dermally. In some embodiments, the biosensor may be implanted in a subject with transdermal microbeads, wherein the optical signals can be transmitted remotely between the biosensor and detecting device.
[0404] In some embodiments, the biosensor is administered as an optode. As used herein, “optode” refers to an optical fiber with a single biosensor, or a composite biosensor, immobilized at the surface or at the end. An “optode” may also be referred to as an “optrode.” In some embodiments, the biosensor is implanted in a subject as an optode. The optode may be incorporated with or into a needle. The optode may be incorporated with a probe such as endoscopy or colonoscopy probes. The optode may be used in a tumor, near a tumor, or at the periphery of a tumor. In some embodiments, the biosensor may be implanted in a subject as an optode, wherein the optical signals can be transmitted between the biosensor and detecting device using physical links. In some embodiments, the biosensor is administered as an optode to a sample or reaction. The optode may be contacted with a sample or reaction. In some embodiments, an optode is used to continuously or episodically monitor a ligand in a sample or reaction.
Methods of Detecting the Presence of a Ligand
[0405] Provided herein is a method of detecting the presence of a ligand in a sample. The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a ligand-free control. A difference in signal indicates the presence of ligand in the sample.
[0406] Also provided herein is a method of detecting the presence of glucose in a sample. The method may include (a) providing a glucose biosensor disclosed herein in which the reporter group is attached the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to glucose present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with the signal transduced by the reporter group when the biosensor is contacted with a glucose-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the test sample, as compared to when the biosensor is contacted with the control sample, indicates that the test sample contains glucose.
Methods of Determining the Concentration of a Ligand
[0407] Provided herein is a method of determining the concentration of a ligand in a sample. The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
[0408] Another aspect of the present disclosure provides a method of determining the concentration of glucose in a test sample comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor comprising a glucose biosensor as described herein in which the reporter group is attached the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to glucose present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with a standard, e.g., hyperbolic glucose binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of glucose to determine the concentration of glucose in the test sample.
Methods of Monitoring the Presence of a Ligand
[0409] The present invention is directed to a method of episodically or continuously monitoring the presence of a ligand in a reaction. In certain embodiments, the biosensors may be used in the continuous monitoring of glucose in a reaction. In certain embodiments, the glucose sensors may be used in episodic monitoring of sample aliquots.
[0410] The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the bio sensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; and episodically or continuously monitoring the signal from the biosensor in the reaction.
[0411] The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the bio sensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; episodically or continuously monitoring the signal from the biosensor in the reaction; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
[0412] In some embodiments, the method further includes comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the reaction.
[0413] In some embodiments, the method further includes comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
[0414] Another aspect of the present disclosure provides a method of continuously monitoring the presence of glucose in a reaction comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor as described herein in which the reporter group is attached the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose; (b) maintaining the biosensor within the reaction and under conditions such that the biosensor can bind to glucose present in the reaction; (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction; and optionally (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction with the signal transduced by the reporter group when the biosensor is contacted with a glucose-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction, as compared to when the biosensor is contacted with the control sample, indicates glucose is present in the reaction.
[0415] Yet another aspect of the present disclosure provides a method of continuously monitoring the concentration of glucose in a reaction comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor comprising a glucose biosensor as described herein in which the reporter group is attached the glucose-binding protein so that a signal transduced by the reporter group when the glucose-binding protein is bound to glucose differs from a signal transduced by the reporter group when the glucose-binding protein is not bound to glucose; (b) maintaining the biosensor within the reaction under conditions such that the biosensor can bind to glucose present in the reaction; and (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction; and (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction with a standard hyperbolic glucose binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of glucose to determine the concentration of glucose in the reaction.
General Definitions
[0416] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
[0417] As used herein, the term “about” in the context of a numerical value or range means±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
[0418] In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible
[0419] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
[0420] A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
[0421] As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes/nucleic acids or sequences/amino acids that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
[0422] Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
[0423] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
[0424] “Subject” as used herein refers to any organism from which a biological sample is obtained. For example, the sample is a biological fluid or tissue. For example, a subject is one who wants or is in need of detecting ligand or determining the concentration of ligand with the herein described biosensors. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant.
[0425] As used herein, an “expression vector” is a DNA or RNA vector that is capable of effecting expression of one or more polynucleotides. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically include plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in host cells of the present invention, including in one of the prokaryotic or eukaryotic cells described herein, e.g., gram-positive, gram-negative, pathogenic, non-pathogenic, commensal, cocci, bacillus, or spiral-shaped bacterial cells; archaeal cells; or protozoan, algal, fungi, yeast, plant, animal, vertebrate, invertebrate, arthropod, mammalian, rodent, primate, or human cells. Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of a polynucleotide. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
[0426] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease,” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
[0427] As used herein, “pharmaceutically acceptable” carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.
[0428] The term “diagnosis” refers to a determination that a disease is present in the subject. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop a disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.).
[0429] Unless required otherwise by context, the terms “polypeptide” and “protein” are used interchangeably.
[0430] A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. A variant of any of genes or gene products disclosed herein may have, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof. Variants as disclosed herein also include homologs, orthologs, or paralogs of the genes or gene products described herein. In some embodiments, variants may demonstrate a percentage of homology or identity, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, e.g., in a functional domain, e.g. a ligand-binding or catalytic domain.
[0431] For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using BLAST. For the BLAST searches, the following parameters were employed: (1) Expect threshold is 10; (2) Gap cost is Existence:11 and Extension:1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.”
[0432] The present invention also provides for functional fragments of the genes or gene products described herein. A fragment of a protein is characterized by a length (number of amino acids) that is less than the length of the full length mature form of the protein. A fragment, in the case of these sequences and all others provided herein, may be a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.
[0433] For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350, 350 to 400 amino acids, or 400 to 425 amino acids. The fragments encompassed in the present subject matter comprise fragments that retain functional fragments. As such, the fragments preferably retain the binding domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the binding activity of the protein can be determined.
[0434] As used herein a “biologically active” fragment is a portion of a polypeptide which maintains an activity of a full-length reference polypeptide. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.
[0435] Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired activity and/or specificity.
[0436] Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, glucose binding activity.
[0437] In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
[0438] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. In some embodiments, a mutated or modified protein does not comprise any deletions or insertions. In various embodiments, a mutated or modified protein has less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 deleted or inserted amino acids.
[0439] Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Sites may be substituted in a relatively conservative manner in order to maintain activity and/or specificity. Such conservative substitutions are shown in the table below under the heading of “exemplary substitutions.”
[0440] In certain embodiments, a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in the table below. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.
Exemplary Substitutions
[0441]
TABLE-US-00004 Original Residue Exemplary Substitutions Alanine (Ala) Val; Leu; Ile; Gly Arginine (Arg) Lys Asparagine (Asn) Gln; His Cysteine (Cys) Ser Glutamine (Gln) Asn; His Glutamic Acid (Glu) Asp Glycine (Gly) Pro; Ala Histidine (His) Asn; Gln Isoleucine (Ile) Leu; Val; Ala Leucine (Leu) Ile; Val; Met; Ala; Phe Lysine (Lys) Arg Methionine (Met) Leu; Phe Phenylalanine (Phe) Leu; Val; Ala Proline (Pro) Gly Serine (Ser) Thr Threonine (Thr) Ser Tryptophan (Trp) Tyr Tyrosine (Tyr) Trp; Phe Valine (Val) Ile; Leu; Met; Phe; Ala
[0442] Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity/specificity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for binding a ligand and/or signal transduction.
[0443] In various embodiments, a polypeptide comprises mutations such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is substituted with a cysteine and/or a lysine.
[0444] Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art. In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein.
Key to the Sequence Listing
[0445]
TABLE-US-00005 SEQ ID NO Sequence Name 1 ttGBP1 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_004303.1 and WP_011172778.1] 2 tsGBP2 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_004202647.1 and WP_015717367.1] 3 dmGBP3 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_004171760.1 and WP_013557600.1] 4 tnGBP4 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_002534202.1 and WP_015919155.1] 5 koGBP5 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_002941687.1 and WP_015869326.1] 6 bhGBP6 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. NP_244712.1 and WP_010899970.1] 7 smGBP7 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_001041152.1 and WP_011839435.1] 8 asGBP8 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_831349.1 and WP_011691715.1] 9 ttGBP1 (with signal peptide replaced with M and a HHHHHH at C-terminus) 10 tsGBP2 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 11 dmGBP3 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 12 tnGBP4 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 13 koGBP5 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 14 bhGBP6 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 15 smGBP7 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 16 asGBP8 (with signal peptide replaced with M and a GGSHHHHHH at C- terminus) 17 tsGBP2_C8 (8C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 18 tsGBP2_C9 (9C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 19 tsGBP2_C12 (12C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 20 tsGBP2_C13 (13C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 21 tsGBP2_C41 (41C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 22 tsGBP2_C42 (42C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 23 tsGBP2_C64 (64C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 24 tsGBP2_C66 (66C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 25 tsGBP2_C119 (119C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 26 tsGBP2_C167 (167C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 27 tsGBP2_C223 (223C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 28 tsGBP2_C224 (224C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 29 tsGBP2_C225 (225C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 30 tsGBP2_C244 (244C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 31 tsGBP2_C277 (cysteine substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 32 tsGBP2_C278 (278C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 33 tsGBP2_C312 (312C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 34 tsGBP2_C337 (337C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 35 tsGBP2_C348 (348C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 36 tsGBP2_C357 (357C substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 37 tsGBP2.13C.W8F (13C, 8F double substitution mutant) 38 tsGBP2.13C.W8M (13C, 8M double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 39 tsGBP2.13C.W8Y (13C, 8Y double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 40 tsGBP2.13C.W9F (13C 9F double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 41 tsGBP2.13C.W9M (13C 9M double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 42 tsGBP2.13C.W9Y (13C, 9Y double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 43 tsGBP2.13C.Q64N (13C, 64N double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 44 tsGBP2.13C.Q64E (13C, 64E double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 45 tsGBP2.13C.Q64M (13C, 64M double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 46 tsGBP2.13C.H66Q (13C, 66Q double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 47 tsGBP2.13C.W244M (13C, 244M double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 48 tsGBP2.13C.W244F (13C, 244F double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 49 tsGBP2.13C.W244Y (13C, 244Y double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 50 tsGBP2.13C.D278N (13C, 278N double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 51 tsGBP2.13C.D278S (13C, 278S double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 52 tsGBP2.13C.D278L (13C, 278L double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 53 tsGBP2.13C.K312M (13C, 312M double substitution mutant with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 54 tsGBP2.13C.bZif (13C substitution mutant, with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) 55 tsGBP2.244C.bZif (244C substitution mutant, with bZif fusion with signal peptide replaced with M and a GGSHHHHHH at C-terminus) 56 tsGBP2.13C_244F.bZif (13C, 244F double substitution mutant, with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) 57 Exemplary ttGBP1 expression sequence, optimized using OrfOpt 58 Exemplary tsGBP2 expression sequence, optimized using OrfOpt 59 Exemplary dmGBP3 expression sequence, optimized using OrfOpt 60 Exemplary tnGBP4 expression sequence, optimized using OrfOpt 61 Exemplary koGBP5 expression sequence, optimized using OrfOpt 62 Exemplary bhGBP6 expression sequence, optimized using OrfOpt 63 Exemplary smGBP7 expression sequence, optimized using OrfOpt 64 Exemplary asGBP8 expression sequence, optimized using OrfOpt 65 Exemplary tsGBP2_C8 expression sequence, optimized using OrfOpt 66 Exemplary tsGBP2_C9 expression sequence, optimized using OrfOpt 67 Exemplary tsGBP2_C12 expression sequence, optimized using OrfOpt 68 Exemplary tsGBP2_C13 expression sequence, optimized using OrfOpt 69 Exemplary tsGBP2_C41 expression sequence, optimized using OrfOpt 70 Exemplary tsGBP2_C42 expression sequence, optimized using OrfOpt 71 Exemplary tsGBP2_C64 expression sequence, optimized using OrfOpt 72 Exemplary tsGBP2_C66 expression sequence, optimized using OrfOpt 73 Exemplary tsGBP2_C119 expression sequence, optimized using OrfOpt 74 Exemplary tsGBP2_C167 expression sequence, optimized using OrfOpt 75 Exemplary tsGBP2_C223 expression sequence, optimized using OrfOpt 76 Exemplary tsGBP2_C224 expression sequence, optimized using OrfOpt 77 Exemplary tsGBP2_C225 expression sequence, optimized using OrfOpt 78 Exemplary tsGBP2_C244 expression sequence, optimized using OrfOpt 79 Exemplary tsGBP2_C277 expression sequence, optimized using OrfOpt 80 Exemplary tsGBP2_C278 expression sequence, optimized using OrfOpt 81 Exemplary tsGBP2_C312 expression sequence, optimized using OrfOpt 82 Exemplary tsGBP2_C337 expression sequence, optimized using OrfOpt 83 Exemplary tsGBP2_C348 expression sequence, optimized using OrfOpt 84 Exemplary tsGBP2_C357 expression sequence, optimized using OrfOpt 85 Exemplary tsGBP2.13C.W8F expression sequence, optimized using OrfOpt 86 Exemplary tsGBP2.13C.W8M expression sequence, optimized using OrfOpt 87 Exemplary tsGBP2.13C.W8Y expression sequence, optimized using OrfOpt 88 Exemplary tsGBP2.13C.W9F expression sequence, optimized using OrfOpt 89 Exemplary tsGBP2.13C.W9M expression sequence, optimized using OrfOpt 90 Exemplary tsGBP2.13C.W9Y expression sequence, optimized using OrfOpt 91 Exemplary tsGBP2.13C.Q64N expression sequence, optimized using OrfOpt 92 Exemplary tsGBP2.13C.Q64E expression sequence, optimized using OrfOpt 93 Exemplary tsGBP2.13C.Q64M expression sequence, optimized using OrfOpt 94 Exemplary tsGBP2.13C.H66Q expression sequence, optimized using OrfOpt 95 Exemplary tsGBP2.13C.W244M expression sequence, optimized using OrfOpt 96 Exemplary tsGBP2.13C.W244F expression sequence, optimized using OrfOpt 97 Exemplary tsGBP2.13C.W244Y expression sequence, optimized using OrfOpt 98 Exemplary tsGBP2.13C.D278N expression sequence, optimized using OrfOpt 99 Exemplary tsGBP2.13C.D278S expression sequence, optimized using OrfOpt 100 Exemplary tsGBP2.13C.D278L expression sequence, optimized using OrfOpt 101 Exemplary tsGBP2.13C.K312M expression sequence, optimized using OrfOpt 102 Exemplary tsGBP2.13C.bZif expression sequence, optimized using OrfOpt 103 Exemplary tsGBP2.244C.bZif expression sequence, optimized using OrfOpt 104 Exemplary tsGBP2.13C_244F.bZif expression sequence, optimized using OrfOpt 105 βZif 106 ZF-QNK 107 Hexahistidine Tag 108 Hexalysine Tag 109 ttGBP1 (with signal peptide replaced with M) 110 tsGBP2 (with signal peptide replaced with M) 111 dmGBP3 (with signal peptide replaced with M) 112 tnGBP4 (with signal peptide replaced with M) 113 koGBP5 (with signal peptide replaced with M) 114 bhGBP6 (with signal peptide replaced with M) 115 smGBP7 (with signal peptide replaced with M) 116 asGBP8 (with signal peptide replaced with M) 117 ecGGBP (with signal peptide removed) 118 ttGGBP (NCBI Accession Nos. YP_003852930.1 and WP_013298803.1) 119 stGGBP (NCBI Accession No. WP_001036943.1) 120 chyGGBP (NCBI Accession Nos. WP_013402088.1 and YP_003991244.1) 121 cobGGBP (NCBI Accession Nos. WP_013289482.1 and YP_003839461.1) 122 pspGGBP (NCBI Accession Nos. WP_015735911.1 and YP_003243743.1) 123 csaGGBP (NCBI Accession Nos. WP_013273028.1 and YP_003822565.1) 124 bprGGBP (NCBI Accession Nos. WP_013280279.1 and YP_003830205.1) 125 rinGGBP_A (NCBI Accession Nos. WP_006855636.1 and YP_007778116.1) 126 fprGGBP (NCBI Accession Nos. WP_015536639.1 and YP_007799070.1) 127 cljGGBP (NCBI Accession No. CLJU_c08950) 128 cauGGBP (NCBI Accession No. CAETHG_2989) 129 rinGGBP_B (NCBI Accession Nos. WP_006855628.1 and YP_007778124.1) 130 erhGGBP (NCBI Accession Nos. WP_003775352.1 and YP_004561181.1) 131 ereGGBP (NCBI Accession Nos. WP_012741392.1 and YP_002936409.1) 132 GGSHHHHHH 133 WWXXXXE (conserved sequence) 134 WWXXXE (conserved sequence) 135 XQVXH (conserved sequence) 136 HRXNV (conserved sequence) 137 GDWX (conserved sequence) 138 DXFXXP (conserved sequence) 139 KGSIXA (conserved sequence) 140 ecTrx 141 Adaptor0 142 Adaptor1.0 143 Adaptor2.0a 144 Adaptor2.0b 145 Adaptor3.0 146 Adaptor4.0 147 Adaptor5.0 148 Adaptor6.0 149 Adaptor7.0 150 Adaptor8.0 151 Adaptor9.0 152 Adaptor10.0 153 Adaptor11.0 154 Adaptor12.0 155 Adaptor13 .0 156 Adaptor14.0 157 Adaptor15.0 158 Adaptor16.0
[0446] The terms “bZif” and “βZif” are used synonymously herein.
[0447] Exemplary amino acid sequences are listed below for convenience.
TABLE-US-00006 ttGBP1 (SEQ ID NO: 9) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNARAVL KTRMLGGDPPDTFQVHAGMELIGTWVVANRMEDLSALFRQEGWLQAFPKG LIDLISYKGGIWSVPVNIHRSNVMWYLPAKLKGWGVNPPRTWDKFLATAQ TLKQKGLEAPLALGENWTQQHLWESVALAVLGPDDWNNLWNGKLKFTDPK AVRAWEVFGRVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMT TTLKLKPGTDFAWAPSPGTQGVFMMLSDSFGLPKGAKNRQNAINWLRLVG SKEGQDTSNPLKGSIAARLDSDPSKYNAYGQSAMRDWRSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQTRNPQAAANAAQAIADQVGLGRLGQHHHHHH ** tsGBP2 (SEQ ID NO: 10) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH dmGBP3 (SEQ ID NO: 11) MKLEIFSWWSGDEGPALEALVKLYKQKYPSVDVVNATVAGGAGTNAKAVL KTRMLGGDPPDSFQAHAGQELIGTWVVANRMEDLSSLFKSEGWTTKFPKD LLPLISSKGGIWSVPVNVHRSNVMWYIPANLKKWGVTAPKTWDQFLTTAK TLKTKNVTPLALGENWTQQHLWESVAVGTLGAQGWQNLWSGKLKFTDPKV VKVWDTFGKVLDATNKDASGLSWQQATDRVVNGQAAFNIMGDWAAGYLST TKKLKPGTGFGWAPSPSTSGTFIFLADSFGLPKGAKDRAEALSWLKLLGS KQGQDTFNPLKGSIAARVDSDLSKYSTYSQSAAKDWKSNKIVGSLTHGAV APESFTSTFGTVIDAFVASRNAQVAAATTQQLADKAGLGKGGSHHHHHH tnGBP4 (SEQ ID NO: 12) MLEIFSWWTAGGEAEALEALIKVFNKYYPDVEVINATVAGGAGTNAKAVL KTRILGGNPPDSFQVHAGMELIDTYVIPGYMTPITNLLEQWGVMDKFPKG ILEMASYEGEIYSIPVNVHRGNVVFYNKKIAEEIGMNEPPKTWDEFIMYL QKAKEKGYVGLALGDKNKWTALHLFETILLGVLGPNDYNGLWKGEVSFND PRIRRAFEIMNKLLDYVNEDHAALAWQDATRLVYEGKALANVMGDWAEGY LKSVGWEPGKDFGWFAVPETQNAFMVVSDTFGLPKNAPHKENAVKWLKVV ASVEGQDAFNPIKGSIPARLDADRSKYDIYLQWSMEDFATKALTPSIAHG SAAPEGFVTTLNDIINRFVTTRDIDSALEELLMAAEDEGYLVEGGSHHHH HH koGBP5 (SEQ ID NO: 13) MLEIFSWWTGGGEEEGLLALFDVFHKYYPDVEIINATVAGGAGTNAKAVL KTRMLGGNPPDSFQVHGGMELIDTYVVTGMMEPITDLLEEWGIIDKFPED ILKIASYKGEVYSIPVNVHRGNVVFYNKAILEEVGIEKVPSTWPEFIEVL KKIKKAGYIPLALGDKNKWTATHLFEDILLSTLGPYNYNGLWNGRTSFEH QGVKEALEIFKELMNYVNPNHASLTWQDATLLVFEGKAAFNVMGDWAEGY LKTLGWTPGKEFGWMVVPGTKGSFMVVTDTFGLPKNAPHRENAIKWLKII SSVEGQDTFNPIKGSIPARIDADRSLYDDYLIWSMDDFATNALCPSIIHG SAAPEAFVTALNDTINMFITRKDVKKALKEIIYAAEDYLEGGSHHHHHH bhGBP6 (SEQ ID NO: 14) MLEIFSWWTGAGEEDGLLALIELFEEKHPEIEVDNAAVAGGAGTNAKAVL TSRMQGNDPPGTFQVHGGAELNDSWVAAGQMDPLNDLFEAEGWADKFPEE LIELVSKDGNIYSVPVNIHRGNVLWYNTEIFEEHGLEVPTTFEEFFDVAD ALQEAGVTPLALGDREPWAATHLFETVLLGTLGADDYNKLWSGEVGMDDP RVEEAAEIFIRMLDYVNEDHSSRNWQDASQLVAQGEAAMNVMGDWAKGYF VNDLNLAVKEDFGWAATPGTEGTFMVITDTFGLPTGVENPEVVKSFLAVL GSQEGQDAFNPLKGSIPARVDADVSKYDEYGQETIEDFKSAELSPSLAHG SAANEGFLTQVNQAINIFVTQKDVDSFVDSLKQYQPGGSHHHHHH smGBP7 (SEQ ID NO: 15) MELVIYHWWTAGGEREAINAVFQVFKQKYPNIQIVENPVAGGAGSVMKSV IIGLLAAGTPPDTFQVHAGAELKEYVDAGYLAPIDDIWSKLGLDKVIPST LQVMAKFNGHYYAVPIDVHRSNVLWYNPKIFNELGIINKFGDPRNWSVDT LLQVARYIKQQRPDIAPIALASRNKWPVTHLFEVLLANAGGPETYVKFFT GKFNYNDPNDPVVQTVKKVLTVMATMAKEGLFNSNHPELTWDQAAALVAE GKAAMFIHGDWVAGYYIANNYKYGKDWAAAPFPKNIFILLSDAFELPKNA PHPEAAKDWLMVVGSKEAQEKFNLIKGSIPARTDVSPKYPDPYRPETAED FQKSTLIPSAVHGGIAKEAFMTDLHNILTSMLTAVSVGTPVDNAVNTALA QILQSVKTSGLASFWKGYTIDYFITKRGGSHHHHHH asGBP8 (SEQ ID NO: 16) MKLEITSWWTSGSEADALNVLIDGVKAAKPGLSVDNAAVSGGGGANARQA LAARLQAGSPPDAWQVHPAGQLKSYVDGGQVADLTDLWTEGDWASQMPKD VAEAQQVDGKYYTVPIGVHRGNVLWTNPAVLSKANVTIDADAGIDGLISS LEQVQASGTTPLALGDKDIFASSQLLESLIMSRAGADNWTKLFTSEYSFD APEVKQALEDYKTILSFANKDHSAITWDEAAKKMADGEAAVNLMGDWAYG ELLNAGKKPGTDFAWVAFPGKEDIFDYVGDGFSIPANNIPHAEAARAWLK TLMDPKIQTEFAAKKGSIPAVTSADISGLSEYQQEAAKSLASGAVVSSLA HAQAAGAEFAQTYADAVSTFNGSGNTDAFIASMTQAQKTQLGGSHHHHHH tsGBP2 Cysteine Scans tsGBP2_C8 (SEQ ID NO: 17) MKLEIFSCWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C9 (SEQ ID NO: 18) MKLEIFSWCAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C12 (SEQ ID NO: 19) MKLEIFSWWAGCEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C13 (SEQ ID NO: 20) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C41 (SEQ ID NO: 21) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGCAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C42 (SEQ ID NO: 22) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGCGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C64 (SEQ ID NO: 23) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFCVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C66 (SEQ ID NO: 24) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVCAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C119 (SEQ ID NO: 25) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNICRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C167 (SEQ ID NO: 26) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENCTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C223 (SEQ ID NO: 27) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLCWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C224 (SEQ ID NO: 28) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSCQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C225 (SEQ ID NO: 29) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWCQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C244 (SEQ ID NO: 30) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDCAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C277 (SEQ ID NO: 31) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLCDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C278 (SEQ ID NO: 32) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSCSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C312 (SEQ ID NO: 33) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLCGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C337 (SEQ ID NO: 34) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDCKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C348 (SEQ ID NO: 35) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVCGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2_C357 (SEQ ID NO: 36) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFCSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2 13C Affinity Mutants tsGBP2.13C.W8F (SEQ ID NO: 37) MKLEIFSFWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** sGBP2.13C.W8M (SEQ ID NO: 38) MKLEIFSMWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W8Y (SEQ ID NO: 39) MKLEIFSYWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W9F (SEQ ID NO: 40) MKLEIFSWFAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W9M (SEQ ID NO: 41) MKLEIFSWMAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W9Y (SEQ ID NO: 42) MKLEIFSWYAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.Q64N (SEQ ID NO: 43) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFNVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.Q64E (SEQ ID NO: 44) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFEVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.Q64M (SEQ ID NO: 45) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFMVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.H66Q (SEQ ID NO: 46) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVQAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W244M (SEQ ID NO: 47) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDMAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W244F (SEQ ID NO: 48) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDFAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.W244Y (SEQ ID NO: 49) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDYAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.D278N (SEQ ID NO: 50) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSNSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.D278S (SEQ ID NO: 51) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSSSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.D278L (SEQ ID NO: 52) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSLSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** tsGBP2.13C.K312M (SEQ ID NO: 53) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLMGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSHHHHHH ** bZif Fusions tsGBP2.13C.bZif (SEQ ID NO: 54) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDWAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSGGSTGE KPYKCPECGKSFSRSGGSHHHHHH** tsGBP2.244C.bZif (SEQ ID NO: 55) MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDCAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSGGSTGE KPYKCPECGKSFSRSGGSHHHHHH** tsGBP2.13C_244F.bZif (SEQ ID NO: 56) MKLEIFSWWAGDCGPALEALIRLYKQKYPGVEVINATVTGGAGVNAKAVL KTRMLGGDPPDTFQVHAGQELIGTWVVADRMEDLTSLFRQEGWLQAFPKG LIDLLSYKGGIWSVPVNIHRSNVMWYIPAKLKEWGVTPPKTWAEFLATAQ TLKRKGLEAPLALGENWTQQHLWESVALATLGADGWNNLWSGKLKFTDPK AVAVWETFGKVLDAANKDAAGLSWQQAVDRVVQGKAAFNIMGDFAAGYMS TTLKLKPGTDFAWTPSPGTSGIFMMLSDSFGLPKGAKNRQNAINWLKLVG SKEGQDTFNPLKGSIAARLDSDPAKYNAYGQSAMKDWKSNRIVGSLVHGA VAPESFMSQFGTVMEIFLQSRNPQAAANAAQAIANQVGLGRGGSGGSTGE KPYKCPECGKSFSRSGGSHHHHHH**
[0448] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Example 1. Fluorescently Responsive Sensor Engineering Phases
[0449] The engineering of FRSs can be divided into five phases: [0450] 1. Binding protein discovery. A set of glucose-binding protein sequence homologs is identified. Accurate assignment of their ligand-binding function requires application of a prediction method that incorporates information encoded in the experimentally determined three-dimensional structure of known periplasmic glucose-binding proteins. [0451] 2. Experimental lead validation. Synthetic genes are constructed, which are optimized for heterologous expression in Escherichia coli of one or more predicted glucose-binding protein sequences. The glucose-binding properties and thermostabilities of the corresponding expressed, purified proteins are evaluated. [0452] 3. Engineering of fluorescent responses. Semisynthetic fluorescent conjugates of the experimentally validated leads are constructed by first attaching single fluorophores to single cysteine mutants. The effect of glucose binding on the fluorescence emission properties of those conjugates is evaluated. The spectral properties of a subset of responsive fluorophores is improved using a double-labeling strategy in which a second fluorophore is site-specifically attached to a small domain fused to the N- or C-terminus to establish ngmFRET. Those singly or doubly labeled conjugates that evince strong, ratiometric responses are selected as FRS s for use in sensing applications. [0453] 4. Affinity tuning. Single or multiple mutations are introduced by site-directed mutagenesis to alter the glucose-binding affinities of glucose-responsive FRSs. A set of FRS variants is selected that together cover the clinical glucose concentration range with high accuracy. [0454] 5. Device integration. FRSs are immobilized in the sampling component of the analytical device in a manner that preserves their fluorescent response and glucose affinity. Long-term storage conditions are established.
Example 2. Sensor Engineering Phase 1: Identification of a Family of Periplasmic Glucose-Binding Proteins Homologs Using Structurally Assisted Function Evaluation
[0455] As a first step in constructing robust glucose sensor candidates, we examined bacterial genomic sequences to identify periplasmic glucose-binding protein sequences in known (hyper) thermophiles. Homologs from such organisms are likely to encode thermostable proteins. Analysis of enzyme families has shown that overall sequence identity below ˜60% is a weak predictor of function conservation (Todd, 2001, J. Mol. Biol., 307, 1113-1143; Tian, 2003, J. Mol. Biol., 333, 863-882). Furthermore, functional assignments based on sequence homology alone are known to be particularly problematic in the PBP superfamily. For instance, PBPs that by overall sequence identity are predicted to bind oligopeptides were found to bind oligosaccharides. Enzyme functional assignments are improved greatly if a sequence selection filter based on conservation of catalytic residues identified from protein structures is included. Such catalytic residues comprise a subset of all the residues that contact an enzyme substrate or inhibitor. In the case of the PBPs, functional selection filters need to take into account all the protein-ligand contacts that encode the ligand-binding function. Accordingly, we have developed a structurally assisted functional evaluation (SAFE) method to identify PBP sequence homologs with accurately predicted function. The SAFE homolog search method consists of five steps: [0456] 1. Sequence homolog set is collected using the BLAST sequence alignment tool (Altschul et al., 1990, J Mol Biol, 215, 403-10), starting with Thermus thermophilus periplasmic glucose-galactose binding protein (ttGBP1) sequence as a seed. The following BLAST parameters: (1) Expect threshold is 10.0; (2) Gap cost is Existence:11 and Extension:1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.” Permissive settings are used, such that pairwise hits are required to have a minimum of only 20% sequence identity with the seed sequence. The lengths of the hit and seed are mutually constrained such that the alignment covers at least 70% within each partner. This set of sequences defines a universe of possible glucose-binding proteins without accurately assigning function. [0457] 2. Structure-based encoding of biological function. A primary complementary surface comprising the protein residues that form hydrogen bonds and van der Waals contacts with the bound glucose is defined using computer-assisted, visual inspection of the three-dimensional structure of the Thermus thermophilus-glucose complex (Cuneo et al., 2006, J Biol Chem, 284, 33217-23). This definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of glucose-binding proteins within the universe of sequence homologs collected in (1). [0458] 3. Accurate sequence alignment. Tools such as ClustalW (Chenna et al., 2003, Nucleic Acids Res, 31, 3497-500) are used to construct an accurate alignment of all the sequence homologs. The ttGBP1 seed sequence is included in this alignment. This multiple sequence alignment establishes the equivalent positions of the ttGBP1 PCS in each sequence homolog. [0459] 4. Function evaluation. The glucose-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter. A “Hamming distance”, H, is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H>0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode glucose-binding proteins. [0460] 5. Selection of representative SAFE homologs. The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance, (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification.
These steps are encoded in the ProteinHunter software tool, which encodes the flow of execution, applies the PCS search filter, and visualizes the results, and handles organism annotations such as thermophilicity, and Gram stain status.
[0461] The ProteinHunter package always executes BLAST searches, with the following command
[0462] “blastall-p blastp-m 8-b 50000-d % s-i<INPUT FILE>-o<OUTPUT FILE>”
[0463] where <INPUT FILE> and <OUTPUT FILE> specify the input and output files, respectively for a given calculation. This command executes the BLAST alignment program for protein sequences with default parameters, intrinsically set by the program. The BLAST program version is 2.2.24.
[0464] The ProteinHunter package always executes multiple sequence alignments with the following command
[0465] “clustalw-infile=<INPUT FILE>-outfile=<OUTPUTFILE>-align-quiet”
This command executes the CLUSTALW multi-sequence alignment program for protein sequences. There are no user-specified parameter settings that alter the alignment behavior of the program. The CLUSTALW program version is 2.1.
[0466] Annotated genomic and plasmid sequences of 5062 prokaryotes were obtained from the National Center of Biotechnology Information (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/all.gbk.tar.gz). The protein sequence for the Thermus thermophiles glucose-galactose binding protein (ttGBP1) was extracted from the protein structure file 2b3b (Cuneo et al., 2006, J Biol Chem, 284, 33217-23), and used as the seed sequence for the BLAST search described above. A total of 1120 sequence homologs from 736 genomes were identified, of which 140 had PCS residues that satisfied the PCS filter.
[0467] In ttGBP1, glucose binding is encoded by a PCS comprising eleven residues. This PCS consists of four tryptophan residues three of which form hydrogen bonds to either the hydroxyls (W9 and W224) or the pyranose ring (W8), the fourth tryptophan forms extensive van der Waals interactions with the pyranose ring (W244). The other seven residues (E13, Q64, H66, H119, D278, K312, and H348) form hydrogen bonds with all the glucose hydroxyls (
TABLE-US-00007 TABLE 1 Residues in that form the primary complementary surface in ttGBP1.sup.a. Residue Interaction W8 Indole hydrogen bond to center of pyranose ring W9 Indole hydrogen bond with 3-OH E13 Hydrogen bonds with 4-OH and 6-OH Q64 Hydrogen bond with 3-OH H66 N.sub.ε forms hydrogen bond with 2-OH H119 Potential hydrogen bond with 3-OH and 4-OH W224 Indole hydrogen bond with 6-OH W244 Aromatic ring forms extensive van der Waals contacts with pyranose ring D278 Hydrogen bonds with 1-OH and 4-OH K312 Hydrogen bonds with 3-OH and 4-OH H348 No forms hydrogen bond with 1-OH .sup.aSingle-letter amino acid code. Positions based on structure from PDB accession 2b3b.
TABLE-US-00008 TABLE 2 PCS position and sequence Iden- Thermo- # Accession code 8 9 13 64 66 119 224 244 278 312 348 tity philicity Gram Organism 1 NC_005835|YP_004303.1 W W E Q H H W W D K H 0.99 Thermophilic − Thermus thermophilus 2 NC_017587|YP_006059052.1 W W E Q H H W W D K H 0.98 Thermophilic − Thermus thermophilus 3 NC_017272|YP_005640237.1 W W E Q H H W W D K H 0.97 Thermophilic − Thermus thermophilus 4 NC_014974|YP_004202647.1 W W E Q H H W W D K H 0.91 Thermophilic − Thermus scotoductus 5 NC_019386|YP_006972235.1 W W E Q H H W W D K H 0.9 Mesophilic − Thermus oshimai 6 NC_017278|YP_005654113.1 W W E Q H H W W D K H 0.9 Mesophilic − Thermus sp. 7 NC_014212|YP_003685745.1 W W E Q H H W W D K H 0.82 Thermophilic + Meiothermus silvanus 8 NC_013946|YP_003505968.1 W W E Q H H W W D K H 0.82 Thermophilic + Meiothermus ruber 9 NC_019793|YP_007182364.1 W W E Q H H W W D K H 0.75 Mesophilic + Deinococcus peraridilitoris 10 NC_014958|YP_004171760.1 W W E Q H H W W D K H 0.73 Mesophilic + Deinococcus maricopensis 11 NC_012526|YP_002785095.1 W W E Q H H W W D K H 0.7 Mesophilic + Deinococcus deserti 12 NC_008025|YP_604376.1 W W E Q H H W W D K H 0.7 Mesophilic + Deinococcus geothermalis 13 NC_017790|YP_006260352.1 W W E Q H H W W D K H 0.69 Mesophilic + Deinococcus gobiensis 14 NC_014221|YP_003703981.1 W W E Q H H W W D K H 0.68 Thermophilic − Truepera radiovictrix 15 NC_017790|YP_006260037.1 W W E Q H H W W D K H 0.68 Mesophilic + Deinococcus gobiensis 16 NC_014364|YP_003805093.1 W W E Q H H W W D K H 0.65 Mesophilic − Spirochaeta smaragdinae 17 NC_020409|YP_007493932.1 W W E Q H H W W D K H 0.65 Mesophilic − Desulfovibrio piezophilus 18 NC_016803|YP_005168947.1 W W E Q H H W W D K H 0.65 Mesophilic − Desulfovibrio desulfuricans 19 NC_012881|YP_002990109.1 W W E Q H H W W D K H 0.64 Mesophilic − Desulfovibrio salexigens 20 NC_007519|YP_390145.1 W W E Q H H W W D K H 0.62 Mesophilic − Desulfovibrio alaskensis 21 NC_016633|YP_005062569.1 W W E Q H H W W D K H 0.62 Mesophilic + Sphaerochaeta pleomorpha 22 NC_014484|YP_003873938.1 W W E Q H H W W D K H 0.52 Thermophilic − Spirochaeta thermophila 23 NC_017583|YP_006045430.1 W W E Q H H W W D K H 0.51 Thermophilic − Spirochaeta thermophila 24 NC_013525|YP_003321952.1 W W E Q H H W W D K H 0.5 Hyperthermo- + Thermobaculum terrenum philic 25 NC_015707|YP_004660650.1 W W E Q H H W W D K H 0.49 Hyperthermo- − Thermotoga thermarum philic 26 NC_011961|YP_002523752.1 W W E Q H H W W D K H 0.49 Thermophilic − Thermomicrobium roseum 27 NC_011978|YP_002534202.1 W W E Q H H W W D K H 0.49 Hyperthermo- − Thermotoga neapolitana philic 28 NC_011661|Dtur_1808 W W E Q H H W W D K H 0.48 Thermophilic + Dictyoglomus turgidum 29 NC_014960|YP_004172812.1 W W E Q H H W W D K H 0.48 Thermophilic − Anaerolinea thermophila 30 NC_009328|YP_001127216.1 W W E Q H H W W D K H 0.48 Thermophilic + Geobacillus thermodenitrifican 31 NC_022080|M493_16625 W W E Q H H W W D K H 0.48 Thermophilic + Geobacillus sp. 32 NC_016593|YP_004984007.1 W W E Q H H W W D K H 0.47 Mesophilic + Geobacillus thermoleovorans 33 NC_006510|YP_149060.1 W W E Q H H W W D K H 0.47 Thermophilic + Geobacillus kaustophilus 34 NC_017934|YP_006345378.1 W W E Q H H W W D K H 0.47 Mesophilic + Mesotoga prima 35 NC_015387|YP_004368987.1 W W E Q H H W W D K H 0.47 Thermophilic − Marinithermus hydrothermalis 36 NC_013411|YP_003254339.1 W W E Q H H W W D K H 0.47 Thermophilic + Geobacillus sp. 37 NC_011653|YP_002334739.1 W W E Q H H W W D K H 0.47 Thermophilic − Thermosipho africanus 38 NC_009828|YP_001470668.1 W W E Q H H W W D K H 0.47 Hyperthermo- − Thermotoga lettingae philic 39 NC_009848|YP_001488451.1 W W E Q H H W W D K H 0.47 Mesophilic + Bacillus pumilus 40 NC_015660|YP_004586376.1 W W E Q H H W W D K H 0.46 Hyperthermo- + Geobacillus philic thermoglucosidasiu 41 NC_020210|YP_007403540.1 W W E Q H H W W D K H 0.46 Thermophilic + Geobacillus sp. 42 NC_009523|RoseRS_0803 W W E Q H H W W D K H 0.46 Thermophilic − Roseiflexus sp. 43 NC_009616|YP_001306064.1 W W E Q H H W W D K H 0.46 Thermophilic − Thermosipho melanesiensis 44 NC_002570|NP_244712.1 W W E Q H H W W D K H 0.46 Mesophilic + Bacillus halodurans 45 NC_009767|Rcas_1174 W W E Q H H W W D K H 0.46 Thermophilic − Roseiflexus castenholzii 46 NC_012785|YP_002941687.1 W W E Q H H W W D K H 0.46 Mesophilic − Kosmotoga olearia 47 NC_021171|YP_007909332.1 W W E Q H H W W D K H 0.44 Mesophilic + Bacillus sp. 48 NC_010003|YP_001568934.1 W W E Q H H W W D K H 0.44 Thermophilic − Petrotoga mobilis 49 NC_017455|YP_005837174.1 W W E Q H H W W D K H 0.44 Mesophilic − Halanaerobium praevalens 50 NC_016751|YP_005097730.1 W W E Q H H W W D K H 0.44 Mesophilic + Marinitoga piezophila 51 NC_013595|YP_003343338.1 W W E Q H H W W D K H 0.44 Mesophilic + Streptosporangium roseum 52 NC_019978|YP_007315359.1 W W E Q H H W W D K H 0.43 Mesophilic + Halobacteroides halobius 53 NC_018524|YP_006643279.1 W W E Q H H W W D K H 0.43 Mesophilic + Nocardiopsis alba 54 NC_017079|YP_005442135.1 W W E Q H H W W D K H 0.42 Mesophilic + Caldilinea aerophila 55 NC_009953|YP_001538287.1 W W E Q H H W W D K H 0.39 Mesophilic + Salinispora arenicola 56 NC_008699|YP_922504.1 W W E Q H H W W D K H 0.38 Mesophilic + Nocardioides sp. 57 NC_019395|YP_006981831.1 W W E Q H H W W D K H 0.38 Mesophilic + Propionibacterium acidipropion 58 NC_021064|YP_007870224.1 W W E Q H H W W D K H 0.37 Mesophilic + Propionibacterium avidum 59 NC_009380|YP_001160076.1 W W E Q H H W W D K H 0.37 Mesophilic + Salinispora tropica 60 NC_021085|YP_007888188.1 W W E Q H H W W D K H 0.37 Mesophilic + Propionibacterium acnes 61 NC_014830|YP_004097363.1 W W E Q H H W W D K H 0.37 Mesophilic + |ntrasporangium calvum 62 NC_014039|YP_003582156.1 W W E Q H H W W D K H 0.37 Mesophilic + Propionibacterium acnes 63 NC_017803|YP_006269831.1 W W E Q H H W W D K H 0.36 Mesophilic + Actinoplanes sp. 64 NC_018707|YP_006851986.1 W W E Q H H W W D K H 0.36 Mesophilic + Propionibacterium acnes 65 NC_013172|YP_003156024.1 W W E Q H H W W D K H 0.35 Mesophilic + Brachybacterium faecium 66 NC_017093|YP_005466360.1 W W E Q H H W W D K H 0.35 Mesophilic + Actinoplanes missouriensis 67 NC_017550|YP_005985051.1 W W E Q H H W W D K H 0.35 Mesophilic + Propionibacterium acnes 68 NC_013729|YP_003384633.1 W W E Q H H W W D K H 0.35 Mesophilic + Kribbella flavida 69 NC_014246|YP_003718075.1 W W E Q H H W W D K H 0.34 Mesophilic + Mobiluncus curtisii 70 NC_013947|YP_003509686.1 W W E Q H H W W D K H 0.34 Mesophilic + Stackebrandtia nassauensis 71 NC_009033|YP_001041152.1 W W E Q H H W W D K H 0.33 Hyperthermo- N/a Staphylothermus marinus philic 72 NC_014205|YP_003669472.1 W W E Q H H W W D K H 0.33 Hyperthermo- N/a Staphylothermus hellenicus philic 73 NC_014804|YP_004071798.1 W W E Q H H W W D K H 0.32 Hyperthermo- N/a Thermococcus barophilus philic 74 NC_008541|YP_831349.1 W W E Q H H W W D K H 0.32 Mesophilic + Arthrobacter sp. 75 NC_000961|NP_143109.1 W W E Q H H W W D K H 0.31 Hyperthermo- N/a Pyrococcus horikoshii philic 76 NZ_CP006965| W W E Q H H W W D K H 0.3 ? N/a Methanobacterium sp. WP_042682828.1 77 NC_009434|PST_2440 W W E Q K H W W D K H 0.3 Mesophilic − Pseudomonas stutzeri 78 NC_009439|YP_001186649.1 W W E Q K H W W D K H 0.29 Mesophilic − Pseudomonas mendocina 79 NC_021577|M062_17030 W W E Q K H W W D K H 0.29 Mesophilic − Pseudomonas aeruginosa 80 NC_017584|YP_006046361.1 W W E Q K H W W D K H 0.28 Mesophilic − Rhodospirillum rubrum 81 NC_006371|YP_133554.1 W W E Q K H W W D K Q 0.28 Psychrophilic − Photobacterium profundum 82 NC_017986|YP_006387725.1 W W E Q K H W W D K H 0.28 Mesophilic − Pseudomonas putida 83 NC_014532|YP_003898163.1 W W E Q N H W W D K H 0.28 Mesophilic − Halomonas elongata 84 NC_017506|YP_005886650.1 W W E Q K H W W D K H 0.28 ? − Marinobacter adhaerens 85 NC_007645|YP_437156.1 W W E Q K H W W D K H 0.28 Mesophilic − Hahella chejuensis 86 NC_018028|YP_006458250.1 W W E Q K H W W D K H 0.28 Mesophilic − Pseudomonas stutzeri 87 NC_010501|YP_001751058.1 W W E Q K H W W D K H 0.27 Mesophilic − Pseudomonas putida 88 NC_007963|YP_574462.1 W W E Q K H W W D K H 0.27 Mesophilic − Chromohalobacter salexigens 89 NC_014965|YP_004189360.1 W W E Q K H W W D K H 0.27 Mesophilic − Vibrio vulnificus 90 NC_015556|YP_004474128.1 W W E Q K H W W D K H 0.27 Mesophilic − Pseudomonas fulva 91 NC_023064|U771_25180 W W E Q K H W W D K H 0.27 Mesophilic − Pseudomonas sp. 92 NZ_AOIV00000000| W W E Q W H W W D K H 0.27 Mesophilic + Halogeometricum pallidum WP_008383305.1 93 NC_012660|YP_002874357.1 W W E Q K H W W D K H 0.26 Mesophilic − Pseudomonas fluorescens 94 NC_014729|YP_004036449.1 W W E Q W H W W D K H 0.26 Mesophilic + Halogeometricum borinquense 95 NC_022223|N175_10020 W W E Q K H W W D K H 0.26 Mesophilic + Listonella anguillarum 96 NC_004578|NP_791121.1 W W E Q K H W W D K H 0.26 Mesophilic − Pseudomonas syringae 97 NC_018080|YP_006481672.1 W W E Q K H W W D K H 0.26 Mesophilic − Pseudomonas aeruginosa 98 NC_015733|YP_004700499.1 W W E Q K H W W D K H 0.26 Mesophilic − Pseudomonas putida 99 NC_016602|YP_004993577.1 W W E Q K H W W D K H 0.26 Mesophilic − Vibrio furnissii 100 NC_015276|YP_004315089.1 W W E Q K H W W D K H 0.26 Mesophilic − Marinomonas mediterranea 101 NZ_ALJD00000000| W W E Q W H W W D K H 0.25 Mesophilic + Halogranum salarium WP_009367379.1 102 NC_021313|YP_008055550.1 W W E Q W H W W D K H 0.25 Mesophilic + Salinarchaeum sp. 103 NC_023076|X970_03415 W W E Q K H W W D K H 0.25 Mesophilic − Pseudomonas monteilii 104 NC_022738|PVLB_20095 W W E Q K H W W D K H 0.25 Mesophilic − Pseudomonas sp. 105 NC_021505|YP_008115339.1 W W E Q K H W W D K H 0.25 Mesophilic − Pseudomonas putida 106 NZ_AOLZ00000000| W W E Q W H W W D K H 0.25 Mesophilic + Halobiforma lacisalsi WP_007142826.1 107 NZ_AOIL00000000| W W E Q W H W W D K H 0.25 Mesophilic + Natrialba taiwanensis WP_006827663.1 108 NC_008027|YP_609880.1 W W E Q K H W W D K H 0.25 Mesophilic − Pseudomonas entomophila 109 NC_021884|BDL_2837 W W E Q K H W W D K H 0.25 Mesophilic − Burkholderia pseudomallei 110 NZ_AOJI00000000| W W E Q W H W W D K H 0.25 Mesophilic + Halorubrum aidingense WP_008001569.1 111 NC_018643|YP_006756306.1 W W E Q K H W W D K H 0.25 Mesophilic + alpha proteobacterium 112 NC_009080|YP_001081530.1 W W E Q K H W W D K H 0.25 Mesophilic − Burkholderia mallei 113 NC_007908|Rfer_1097 W W E Q K H W W D K Q 0.25 Mesophilic + Albidiferax ferrireducens 114 NZ_AOIP00000000| W W E Q W H W W D K H 0.25 Mesophilic + Natrialba aegyptia WP_006663935.1 115 NC_021173|YP_007917520.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia thailandensis 116 NC_010084|YP_001580612.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia multivorans 117 NZ_AOI000000000| W W E Q W H W W D K H 0.24 Mesophilic + Natrialba asiatica WP_006109396.1 118 NC_011000|YP_002232151.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia cenocepacia 119 NC_017911|YP_006325578.1 W W E Q K H W W D K H 0.24 Mesophilic − Pseudomonas fluorescens 120 NC_019792|YP_007179189.1 W W E Q W H W W D K H 0.24 Mesophilic + Natronobacterium gregoryi 121 NC_010551|YP_001807547.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia ambifaria 122 NZ_AOIB00000000| W W E Q W H W W D K H 0.24 Mesophilic + Natronococcus amylolyticus WP_005559649.1 123 NZ_AOHX00000000| W W E Q W H W W D K H 0.24 Mesophilic + Natronorubrum sulfidifaciens WP_008163842.1 124 NC_017831|YP_006273771.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia pseudomallei 125 NC_004129|YP_261701.1 W W E Q K H W W D K H 0.24 Mesophilic − Pseudomonas protegens 126 NC_007005|YP_234205.1 W W E Q K H W W D K H 0.24 Mesophilic − Pseudomonas syringae 127 NC_006348|YP_103699.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia mallei 128 NC_015379|YP_004352396.1 W W E Q K H W W D K H 0.24 Mesophilic − Pseudomonas brassicacearum 129 NC_017920|YP_006331954.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia sp. 130 NC_010681|YP_001894671.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia phytofirmans 131 NC_009256|YP_001118730.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia vietnamiensis 132 NC_007510|YP_368312.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia lata 133 NC_020209|YP_007398595.1 W W E Q K H W W D K H 0.24 Mesophilic − Pseudomonas poae 134 NC_008390|YP_772721.1 W W E Q K H W W D K H 0.24 Mesophilic − Burkholderia ambifaria 135 NC_010622|YP_001856864.1 W W E Q K H W W D K H 0.23 Mesophilic − Burkholderia phymatum 136 NC_016589|YP_004976536.1 W W E Q K H W W D K H 0.23 Mesophilic − Burkholderia sp. 137 NC_020802|YP_007640421.1 W W E Q K H W W D K Q 0.23 Mesophilic + Psychromonas sp. 138 NC_008687|YP_917171.1 W W E Q K H W W D K H 0.23 Mesophilic − Paracoccus denitrificans 139 NC_014323|YP_003777923.1 W W E Q N H W W D K H 0.22 Mesophilic − Herbaspirillum seropedicae 140 NZ_AOIN00000000| W W E Q W H W W D K H 0.22 Mesophilic + Natrialba chahannaoensis WP_006167401.1
Example 3. Sensor Engineering Phase 2: Lead Protein Validation Using Ligand-Mediated Thermostability Shifts
[0468] Eight homologs that were predicted to be glucose-binding proteins (
[0473] All eight leads produced soluble protein in a T7 expression system in sufficient quantity for functional analysis. The glucose-binding properties of four of these were confirmed directly using the thermal shift assay (Table 3). Four of the GBP homologs exhibited mid-point thermal denaturation temperatures (T.sub.m values) over 100° C. Their glucose-binding properties were verified subsequently using a mutant, fluorescently labeled conjugate that responds to glucose binding (see below).
TABLE-US-00009 TABLE 3 Ligand-binding and thermostability properties of ttGBP1 homologs. Gene NCBI Accession Codes Optimization Soluble Thermostability.sup.d Glucose Name Organism Genome Protein Identity.sup.a Method.sup.b Expression.sup.c .sup.apoT.sub.m (° C.) Binding.sup.e ttGBP1 Thermus thermophilus NC_005835 YP_004303.1 1.0 OrfMorph y >100 y.sup.f tsGBP2 Thermus scotoductus NC_014974 YP_004202647.1 0.91 OrfOpt y >100 y.sup.f dmGBP3 Deinococcus maricopensis NC_014958 YP_004171760.1 0.73 OrfOpt y 47 y tnGBP4 Thermotoga neapolitana NC_011978 YP_002534202.1 0.49 OrfOpt y >100 y.sup.f koGBP5 Kosmotoga olearia NC_012785 YP_002941687.1 0.46 OrfOpt y >100 y.sup.f bhGBP6 Bacillus halodurans NC_002570 NP_244712.1 0.46 OrfOpt y 53 y smGBP7 Staphylothermus marinus NC_009033 YP_001041152.1 0.34 OrfOpt poor 40.sup.f y.sup.f asGBP8 Arthrobacter sp. NC_008541 YP_831349.1 0.32 OrfOpt y 58 y .sup.aNumber of identical residues shared with the probe sequence. .sup.bSee materials and methods. .sup.cJudged by SDS gel electrophoresis of the soluble fraction of a total lysate. .sup.dDetermined in a Roche LightCycler, using SYPRO Orange to monitor the appearance of unfolded protein. .sup.eDetermined by monitoring an increase in the thermostability of the protein in the presence of ligand. .sup.fDetermined using fluorescent Acrylodan and/or Badan conjugates (see text).
[0474] A majority of the sequence identity of these experimentally verified glucose-binding homologs relative the ttGBP1 seed were considerably below the 60% threshold with the sequences identity ranging from 91% to 32%. These results therefore demonstrate that biological function can be predicted accurately with the SAFE technique, even in sequence homologs with low fractional identities to the original seed.
[0475] The homolog from Thermos scotoductus (tsGBP2) was produced at the highest level by heterologous expression in E. coli. This protein was selected as the candidate for constructing robust glucose sensors.
Example 4. Sensor Engineering Phase 3: Cysteine Mutant Scans and Fluorophore Screening to Identify Fluorescently Responsive Glucose Sensors
[0476] Semi-synthetic FRSs can be engineered by site-specifically attaching thiol-reactive, environmentally sensitive fluorophores that respond to ligand-mediated conformational changes. Identification of FRS candidates that can be used for sensing applications comprises three steps: [0477] 1. Cysteine scan. Mutant glucose-binding proteins containing single cysteines are constructed for site-specific attachment of thiol-reactive fluorophores. General structural principles have been established to identify positions in PBPs where attached single fluorophores are likely to exhibit ligand-dependent responses (de Lorimier et al., 2002, Protein Sci, 11, 2655-75). Candidate positions fall into three classes: endosteric, replacing a residue that contacts the ligand directly; peristeric, located at the rim of the binding site; allosteric (Marvin et al., 1997, Proc Natl Acad Sci USA, 94, 4366-71; Marvin, 1998, J Am Chem Soc, 120, 7-11), located outside the binding site at sites that undergo local structural changes in concert with the hinge-bending motion. [0478] 2. Fluorophore screening. Thiol-reactive, environmentally sensitive fluorophores are attached to each cysteine mutant prepared in step 1. [0479] 3. Evaluation of the glucose-mediated change of all the fluorescent conjugates prepared in step 2. Responses to ligand binding in which there is both a change in fluorescence emission intensity and spectral shape are essential for chemometric applications, because such changes enable ratiometric measurements. Changes in spectral shape typically are accompanied by a shift in the wavelength of the emission intensity maxima. Three classes of fluorescent responses are possible: [0480] i. No response. [0481] ii. Monochromatic response (emission intensity increases or decreases without a change in spectral shape) [0482] iii. Dichromatic response (both intensity and spectral shape changes) which can be classified into two sub-classes: [0483] i. Hypsochromatic: emission intensity shifts to shorter wavelengths upon binding glucose (“blue shift”). [0484] ii. Bathochromatic: emission intensity shifts to longer wavelengths upon binding glucose (“red shift”). [0485] 4. Double labeling strategies to convert monochromatic responses into dichromatic signals, or to improve upon dichromatic responses.
[0486] Cysteine scans of tsGBP2. We constructed twenty single cysteine mutants in tsGBP2, exploring thirteen endosteric, five peristeric, and two allosteric positions. At each position we attached the Prodan-derived fluorophores Acrylodan and Badan, which differ by one methylene group in their thiol-reactive linker. The fluorescence emission intensities of twelve Acrylodan and four Badan conjugates responded to glucose at twelve attachment positions (Table 4). At only six attachment positions were the responses of both fluorophores qualitatively similar, and never quantitatively. We also tested for glucose binding by measuring ligand-mediated shifts in protein thermal stability (Table 3).
TABLE-US-00010 TABLE 4 Glucose response of Acrylodan and Badan conjugates in a cysteine scan of the Therm us scotoductus tsGBP2 scaffold. Emission Emission wavelength K.sub.d.sup.c,d,e wavelength K.sub.d.sup.c,d,e Cysteine Fluorophore (nm) (mM) Fluorophore (nm) (mM) position Class.sup.a Cysteine Shape.sup.b λ1 λ2 .sup.appK.sub.d .sup.trueK.sub.d Cysteine Shape.sup.b λ1 λ2 .sup.appK.sub.d .sup.trueK.sub.d W8C e Acrylodan m 462 550 0.008 0.007 Badan m 488 448 70.sup.d 113.sup.d W9C e Acrylodan m 511 461 0.02 0.02 Badan m ns ns D12C p Acrylodan m 513 478 0.005 0.003 Badan m ns ns E13C e Acrylodan d 518 471 0.9 1.1 Badan m 513 571 0.2 0.3 G41C e Acrylodan d 519 474 0.009 0.01 Badan m ns ns A42C e Acrylodan 0 ns ns Badan m ns ns Q64C e Acrylodan 0 ns ns Badan m ns ns H66C e Acrylodan d 486 446 73 155 Badan m ns ns H119C e Acrylodan m 511 461 0.02 0.02 Badan m ns ns W167C p Acrylodan d 492 552 0.02 0.02 Badan m ns ns S223C p Acrylodan 0 ns ns Badan m ns ns W224C e Acrylodan m 483 515 0.7 0.9 Badan 0 ns ns Q225C p Acrylodan m ns ns Badan m ns ns W244C e Acrylodan m 487 450 9.0 19 Badan m 502 452 16 17 S277C a Acrylodan 0 ns ns Badan 0 ns ns D278C e Acrylodan m ns ns Badan 0 ns ns K312C e Acrylodan d 515 465 0.009 0.01 Badan m ns ns W337 a Acrylodan m ns ns Badan 0 ns ns H348 e Acrylodan m 487 515 1.3.sup.d 1.6.sup.d Badan d 523 515 4.3.sup.d 5.2.sup.d M357 p Acrylodan 0 ns ns Badan m ns ns .sup.aa, allosteric e, endosteric; p, peristeric. .sup.bm, monochromatic; d, dichromatic (i.e. spectral shape change); 0, no change. .sup.cns; no or minimal signal change up on glucose addition. .sup.dApproximate values. .sup.eDetermined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equation 1-5.
[0487] Endosteric attachment positions exhibited the most pronounced changes in fluorescence emissions in response to ligand binding. At least one of the two conjugates at all five peristeric positions were responsive to glucose. No allosteric conjugates exhibited fluorescence responses to glucose.
[0488] We observed ligand-dependent shifts in the wavelengths of emission intensity maxima at one peristeric (W167C) and five endosteric (E13C, G41C, H66C, K312C, H348) sites (Table 4), enabling dichromatic ratiometric measurements; the maximum intensity of other glucose-responsive conjugates remained the same (monochromatic responses). Five out of the six positions that enable dichromatic ratiometric measurements were labeled with Acrylodan and the sixth position with Badan. These two fluorophores differ only in their linker geometry, but this small difference determines whether dichromatic or monochromatic responses are observed for a particular conjugate. Changes in linker geometry and chromophore modifications give rise to significant differences in the detailed interactions of particular fluorophores with the protein, even within families of closely related molecules, thereby significantly impacting sensor characteristics, consistent with previous observations.
[0489] In these dichromatic responses of the Acrylodan and Badan conjugates, ligand-mediated changes in emission intensity spectral shapes arise from redistribution of populations of two emission states, ‘blue’ and ‘green’, corresponding to distinct excited state dipoles. Such a redistribution does not occur in monochromatic responses. The emission spectra of all the Acrylodan conjugates undergo a green.fwdarw.blue (hypsochromatic) shift upon ligand binding (Table 4), whereas the emission spectrum of Badan conjugate shifts in the opposite direction (bathochromatic). The Acrylodan conjugate attached to E13C exhibited the largest, wavelength-dependent changes in fluorescence emission intensities.
[0490] Conservation of signaling in glucose-binding protein homologs. The equivalent of the 13C mutation identified in tsGBP2 (see above) was installed in all the other seven ttGBP1 homologs and their Acrylodan and Badan conjugates tested for glucose binding (Table 5). Dichromatic responses were identified in all proteins. In all but one of the proteins, the response of the Acrylodan conjugate was dichromatic, as is the case in tsGBP 13C. The koGBP5 13C Acrylodan conjugate exhibited a monochromatic response, but its Badan conjugate was dichromatic. Both Acrylodan and Badan exhibited dichromatic responses in dmGBP3.
TABLE-US-00011 TABLE 5 ttGBP1 homologs labeled with Acrylodan or Badan.sup.a. Emission Affinity.sup.a,d (nm) (mM) Protein Mutation Conjugate.sup.b Shape.sup.c λ.sub.1 λ.sub.2 .sup.appK.sub.d .sup.trueK.sub.d ttGBP1 E13C A d 486 519 1.9 1.2 B m 496 530 0.5 0.5 tsGBP2 E13C A d 518 471 0.9 1.1 B m 513 571 0.2 0.3 dmGBP3 E13C A d 486 519 7.9 4.9 B d 523 550 0.6.sup.e 0.7.sup.e tnGBP4 E13C A d 487 519 0.73 0.71 B m 527 555 0.096 0.16 koGBP5 E13C A m 491 517 2.4 1.6 B d 535 503 0.2 0.2 bhGBP6 E13C A d 486 515 0.49 0.48 B m 515 490 1.8 1.5 smGBP7 E14C A d 484 463 0.043 0.056 B m 519 490 0.4 0.5 .sup.aDetermined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equation 1-5. .sup.bA, Acrylodan; B, Badan. .sup.cm, monochromatic; d, dichromatic (i.e. spectral shape change); 0, no change. .sup.dnb, no binding. .sup.eApproximate value.
[0491] These results demonstrate that the site of a cysteine mutation that exhibits dichromatic signaling is conserved among homologs. Identification of such a site in one homolog therefore is predictive throughout its protein family identified by the SAFE search method, even for family members that have low sequence identity (e.g. compare ttGBP1 and smGBP7).
[0492] Improving the fluorescence response to glucose in doubly labeled proteins. We tested whether fluorescence energy transfer (FRET) effects in doubly labeled proteins could improve ratiometric signaling. To this end, we fused a small, disulfide-containing domain, βZif (Smith et al., 2005, Protein Sci, 14, 64-73) to the C-terminus of several tsGBP2 cysteine mutants (Table 6). This arrangement enables independent, site-specific labeling with two different, thiol-reactive fluorophores by first reacting at the unprotected thiol in tsGBP2, followed by a reduction of the βZif disulfide to deprotect and label this second site with a second fluorophore. The first fluorophore, attached to tsGBP2 responds directly to glucose binding (directly responsive partner), whereas the second one, attached to the βZif fusion, does not (indirectly responsive partner). Indirectly responsive partners are selected according to their excitation and emission characteristics such that ngmFRET is established with the directly responsive partner. Under favorable circumstances, monochromatic responses of the directly responsive partner or weak dichromatic responses can be converted in to strong ratiometric signals, by exploiting ligand-induced modulation of non-geometrical factors affecting ngmFRET such as changes in spectral overlap between the two partnered fluorophores, and alteration of non-radiative decay rates in the directly responsive partner. Mechanisms for non-geometrically modulated FRET (ngmFRET) effects are detailed in Materials and Methods and PCT International Patent Application No. PCT/US16/62958, filed Nov. 19, 2016, the entire content of which is incorporated herein by reference.
TABLE-US-00012 TABLE 6 Glucose affinities of tsGBP2-βZif fusion proteins.sup.a. Emission Fluorophore wavelength Kd (single Fluorophore (nm) (mM) Construct cysteine) (βZif) λ1 λ2 .sup.appK.sub.d .sup.trueK.sub.d 13C.Acrylodan_βZif.Alexa532 Acrylodan Alexa532 515 548 0.5.sup.b 0.7.sup.b 13C.Acrylodan_βZif.Alexa555 Acrylodan Alexa555 491 556 1.1 1.0 13C.Acrylodan_βZif.TexasRed Acrylodan Texas Red 515 615 0.9 1.2 244C.Acrylodan_βZif.Alexa532 Acrylodan Alexa532 491 545 42 52 244C.Acrylodan_βZif.Alexa555 Acrylodan Alexa555 491 565 17 22 244C.Acrylodan_βZif.TexasRed Acrylodan Texas Red 491 613 14 18 13C 244F.Acrylodan_βZif.Alexa532 Acrylodan Alexa532 519 493 5.3 5.9 13C 244F.Acrylodan_βZif.Alexa555 Acrylodan Alexa532 515 493 4.5 4.8 13C 244F.Acrylodan_βZif.TexasRed Acrylodan Texas Red 519 614 7.7 6.9 .sup.aDetermined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equations 1-5. .sup.bApproximate value.
[0493] The Acrylodan conjugate attached to 244C elicits a strong monochromatic response (Table 4). To test whether this response could be converted into a dichromatic one, we partnered this conjugate with indirectly responsive acceptors Alexa532, Alexa555, and Texas Red, placed on the βZif domain (Table 6). In all cases ngmFRET was established between the two partners, and dichromatic responses were obtained. The wavelength interval for measuring the directly responsive donor intensity was centered near the Acrylodan emission peak, whereas that of the acceptors was placed at the emission maximum of each acceptor. For each of the three conjugates the intensities of both the directly responsive donor and the indirectly responsive acceptor increased with addition of glucose. This is consistent with a mechanism in which the glucose decreases the degree of quenching in the donor without a change in the shape of its emission spectrum, leading to increases in both the radiative emission and energy transfer rates (model d.sup.−ϕ.sup.0, Table 7). The unequal increases in donor and acceptor emission intensities results in dichromatic signals suitable for ratiometry. Alexa532 was the brightest of the three acceptors, and therefore well for glucose sensing.
TABLE-US-00013 TABLE 7 Qualitative analysis of the patterns of donor and acceptor emission intensity changes in ngmFRET.sup.a Directly responsive partner Model Q.sub.A/Q.sub.D Q.sub.D Q.sub.A Donor d.sup.0 ϕ.sup.+ ↑ ↓ ↑ d.sup.0 ϕ.sup.− ↓ ↑ ↓ d.sup.+ ϕ.sup.0 ↓ ↓ ↓ d.sup.+ ϕ.sup.+ * ↓ * d.sup.+ ϕ.sup.− ↓ * ↓ d.sup.− ϕ.sup.0 ↑ ↑ ↑ d.sup.− ϕ.sup.+ ↑ * ↑ d.sup.− ϕ.sup.− * ↑ * Acceptor a.sup.0 ϕ.sup.+ ↑ ↓ * a.sup.0 ϕ.sup.− ↓ ↑ * a.sup.+ ϕ.sup.0 ↓ 0 ↓ a.sup.+ ϕ.sup.+ * ↓ * a.sup.+ ϕ.sup.− ↓ ↑ * a.sup.− ϕ.sup.0 ↑ 0 ↑ a.sup.− ϕ.sup.+ ↑ ↓ ↑ a.sup.− ϕ.sup.− * ↑ * .sup.aThe effects of increasing or decreasing quenching in the directly responsive ngmFRET partner (d for donors, a for acceptors) or the energy transfer coupling (ϕ) between the donor and acceptor are tabulated. The consequences of using a directly responsive donor or acceptor are examined. Changes in quenching and energy transfer coupling parameters can occur singly or in combination, leading to 16 possible models. The models examine the effects of the direction of change in quenching parameters (no change, d.sup.0 or a.sup.0; increase d.sup.+ or a.sup.+; decrease, d.sup.− or a.sup.−) and the energy transfer coupling factor (no change, ϕ.sup.0; increase, ϕ.sup.+; decrease, ϕ.sup.−) on the patterns in the direction of change of the donor, Q.sub.D (equation 30) or acceptor, Q.sub.A (equation 32) quantum yields, and their ratio, Q.sub.A/Q.sub.D (equation 33): ↑, increase; ↓, decrease; 0, no change; *, response is dependent on precise quantitation rather than direction of change in the underlying parameter values.
[0494] We also tested whether the strong dichromatic response observed for the 13C⋅Acrylodan conjugate could be improved upon further by ngmFRET. This conjugate was paired with the three fluorophores described. Energy transfer was established in all three doubly labeled conjugates. The directly responsive donor emission intensity was measured for the blue state, and the three acceptor emissions were measured as described above. In all three cases, the ratio of the acceptor/donor intensities decreased with addition of glucose, as did the directly responsive donor intensities. The indirectly responsive acceptor intensity of the Alexa532 and Texas Red conjugates increased, whereas it decreased for Alexa555. These results are consistent with a mechanism in which the directly responsive Acrylodan donor switches from a green to a blue state, altering the energy transfer coupling factor, 4.
Example 5. Sensor Engineering Phase 4: Affinity Tuning
[0495] Blood glucose concentrations range from ˜3 mM (hypoglycemia) to ˜30 mM (hyperglycemia) and up to ˜100 mM for the hyperosmolar hyperglycemic state (HHS) (Pasquel, 2014, Diabetes Care, 37, 3124-3131), with healthy levels at around 6 mM (euglycemia) (American Diabetes Association, 2000, Clinical Diabetes, 18). Measurements using reagentless sensors are most sensitive at analyte concentrations that match the dissociation constant (de Lorimier et al., 2002, Protein Sci, 11, 2655-75; Marvin et al., 1997, Proc Natl Acad Sci USA, 94, 4366-71). The glucose affinity of tsGBP13C⋅Acrylodan is too high and must therefore be “tuned” by raising the K.sub.d value.
[0496] The mutations that alter glucose affinities can fall into four classes: [0497] 1. Alteration of direct interactions in the PCS between the protein and the bound glucose. [0498] 2. Manipulation of the equilibrium between the open (ligand-free) and closed (ligand-bound) states. [0499] 3. Indirect interactions that alter the geometry of the binding site. [0500] 4. Alteration of interactions between the protein and the fluorescent conjugate.
[0501] Representatives of mutant class 1 were constructed in the tsGBP13C background, using Acrylodan and Badan conjugates to evaluate their effects on glucose binding (
TABLE-US-00014 TABLE 8 Glucose affinities of tsGBP 13C .Math. Acrylodan and Badan conjugates. Emission Glucose wavelength affinity (nm) (mM).sup.c,d Mutation Fluorophore.sup.a Change.sup.b λ.sub.1 λ.sub.2 .sup.appK.sub.d .sup.trueK.sub.d A d 515 481 1.21 1.12 B m/d 513 571 0.23 0.29 W8F A 0 B 0 W8M A d 491 510 0.6.sup.c 0.6.sup.c B m 546 491 0.27 0.38 W8Y A m/d.sup.c B 0 W9F A m 491 466 0.6.sup.c 0.7.sup.c B 0 W9M A m 491 515 0.4.sup.c 0.4.sup.c B m.sup.c W9Y A d 515 495 3.3 2.2 B m 532 487 0.65 1.2 Q64N A d 491 555 10 9.2 B D 515 560 1.3 1.2 Q64E A d 515 555 0.4.sup.c 0.3.sup.c B d 519 555 2.1 2.9 Q64M A m/d 491 555 1.1 0.81 B m 519 492 0.1.sup.c 0.1.sup.c H66Q A d 491 515 20.sup.c 7.sup.c B m 485 540 0.1.sup.c 0.09.sup.c W244M A d 515 490 40.sup.c 20.sup.c B d 527 555 5.sup.c 7.sup.c W244F A d 519 488 4.6 4.6 B d 523 555 2.sup.c 2.sup.c W244Y A d 519 474 5.6 7.8 B d 519 555 4.1 3.8 D278N A d 515 470 100.sup.c 100.sup.c B m/d 523 555 99 120 D278S A m/d 473 556 500.sup.c 300.sup.c B m/d 527 555 81 91 D278L A m 459 549 800.sup.c 400.sup.c B 0 K312M A d 472 554 290 203 B m 527 555 26 39 .sup.aA, Acrylodan; B, Badan. .sup.bm, monochromatic; d, dichromatic (i.e spectral shape change); 0, no-or very small change. .sup.cApproximate value. .sup.dDetermined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equations 1-5.
Example 6. Sensor Arrays for Detecting a Wide Range of Glucose Concentrations
[0502] The precision (reciprocal of the error) of individual sensor precision is maximal at the K.sub.d value, and decreases at lower or higher glucose concentrations (Marvin et al., 1997, Proc Natl Acad Sci USA, 94, 4366-71). Construction of a high-precision sensor capable of spanning the entire 100-fold clinical concentration range from extreme hypoglycemia to the HHS therefore requires combining several sensors together to maintain a high precision level. Candidates include (Tables 4 and 8): tsGBP13C⋅Acrylodan, tsGBP13C⋅Acrylodan 9Y, tsGBP13C⋅Acrylodan 64N, tsGBP13C⋅Acrylodan 66Q, tsGBP13C⋅Acrylodan 244F (Badan), tsGBP13C⋅Acrylodan 244Y (Badan), tsGBP13C⋅Acrylodan 244M (Badan), tsGBP13C⋅Acrylodan 278N. The βZif fusions also can used (Table 6).
Example 7. Sensor Engineering Phase 5: Device Integration
[0503] Protein immobilization on solid surfaces is an important step for incorporating biosensors into devices Immobilization enables (i) spatial localization, (ii) control over the presentation of the sensors to the reader (e.g. by encoding geometries for optical readouts), (iii) selective retention in sample separation procedures. It is advantageous to control the geometry of the protein attachment to the solid surface, in order to minimize perturbation of the fluorescence sensing mechanism. Such constructs fuse an N- or C-terminal protein domain that can mediate site-specific attachment to an appropriately chemically activated surface. For instance, hexa-histidine peptide for metal-mediated immobilization. Here we show that site-specific attachment of a robust glucose sensor to suitably derivatized agarose beads conserves its emission fluorescence spectral response and thermostability.
[0504] The tsGBP13C_244F⋅Acrylodan protein was site-specifically immobilized through its C-terminal hexa-histidine tag on commercially available magnetic beads coated with Ni-NTA. The use of magnetic beads affords a straightforward means for holding the beads in place within their respective sensor patches in the sampling cartridge with a magnetic field. Site-specific immobilization is intended to minimize perturbation of the sensing mechanism. Comparison of protein thermostabilities determined in solution and on beads showed that protein stability was not perturbed by immobilization within the upper limit of the measured temperature (100° C.).
[0505] The magnetic beads coated with immobilized tsGBP13C_244F⋅Acrylodan were dried by incubation at 50° C. for 20 minutes, using an aqueous ammonium bicarbonate buffer. The stability properties of the sensor were recovered upon rehydration in the temperature, as determined up to 100° C. The dried beads were aged in situ inside fully assembled sample-handling cartridges by incubation for up to 7 days at 25° C., 37° C., and 50° C. in the dark. Fluorescence and glucose-responsive properties were tested in cartridges stored for 1, 2 and 7 days. For all drying conditions, the fluorescence ratio in the absence of glucose, and the glucose affinities of the immobilized sensors remained approximately unchanged. The tsGBP2-based FRSs therefore are sufficiently robust to be handled at ambient temperatures in a desiccated state, greatly simplifying manufacturing, distribution, and long-term storage conditions.
Example 8. Materials and Methods
[0506] Bioinformatic searches. Annotated genomic and plasmid sequences of 5062 prokaryotes were obtained from the National Center of Biotechnology Information (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/all.gbk.tar.gz), together with annotations recording prokaryotic lifestyles ( . . . /ProkaryotesOrganismInfo.txt). The Protein Databank (PDB) was obtained from www.rcsb.org. The obtained genomic and structural data files were organized into pre-processed two databases (PG, prokaryotic genomes; PDB). The ‘ProteinHunter’ program provides an interface and methods for organizing, querying, and analyzing these databases. ProteinHunter comprises a graphical user interface, set of computer scripts, and a parallel computing environment. Together these set up the calculations, manage the flow of information and execution in each of the calculation phases, control other programs that carry out specific calculations such as BLAST (Altschul et al., 1990, J Mol Biol, 215, 403-10) and ClustalW (Chenna et al., 2003, Nucleic Acids Res, 31, 3497-500), and visualize the results.
[0507] To construct homolog sequence sets, single sequence seeds were extracted from either preprocessed PDB or PG databases. Homolog sets were then identified in the PDB or PG by using a seed sequence for a uni-directional BLAST search with the following parameters: expect threshold, 10.0; gap costs for existence, 11, and extension, 1; BLOSUM matrix; low complexity filter is on (the ProteinHunter package always executes BLAST searches with the following command “blastall-p blastp-m 8-b 50000-d<database file>-i<input file>-o <output file>, where <database file> specifies the name of the prebuilt search sequence file and <input file> and <output file> the seed sequence input and hit output files respectively. A pairwise BLAST alignment was scored in ProteinHunter as a homolog hit if it exceeded a minimum fraction of identical residues and if the alignment covered at least 70% of the probe and target sequences.
[0508] Function was inferred using the sequence of primary complementary surface (PCS) residues. A 11-residue, non-contiguous sequence comprising the PCS between the protein and the bound glucose in the ttGBP1 structure (PDB entry 2b3b) was identified using ProteinHunter (
[0509] Gene synthesis and mutagenesis. The amino acid sequences for the predicted GBP homologs identified in the bioinformatic search (see above) were extracted from the PG database. The putative leader peptide that mediates anchoring of the periplasmic-binding protein on the outside of the membrane (Gram positive bacteria) or directs secretion into the periplasm (Gram negative bacteria) was deleted by examining the multiple sequence alignment and removing the sequences N-terminal to the start of the mature GBP amino acid sequence. Endogenous cysteines were changed to alanine. A hexahistidine tag was placed behind a GGS linker at the C-terminus of the mature protein to enable metal-mediated affinity purification (Hengen, 1995, Adv Healthc Mater, 2, 43-56). The final amino acid sequences were back-translated into a DNA sequence encoding the open reading frame (ORF), which was placed in a construct behind an efficient Shine-Dalgarno ribosome-binding site, and flanked by a T7 promoter and terminator at the 5′ and 3′ ends respectively, using the GeneFab program (Cox et al., 2007, Protein Sci, 16, 379-90). The resulting ORF sequences were optimized in context by OrfOpt or OrfMorph programs designed to predict highly expressed mRNA sequences in E. coli (see below). The resulting DNA sequences were synthesized by oligonucleotide assembly and cloned into pUC57 by GeneWiz, Inc. (South Plainfield, N.J.).
[0510] Subsequent single and multiple point mutations were designed by preparing mutant sequences of the synthetic ORF sequences using the GfMutagenesis program that introduces point mutations into an ORF using the most prevalent codon in E. coli for an amino acid. Constructs for site-specific double labeling were designed by inserting the βZif domain sequence (Smith et al., 2005, Protein Sci, 14, 64-73) before the hexa-histidine C-terminal purification tag. All variants also were constructed by total gene synthesis.
[0511] Synthetic gene optimization. The OrfOpt program (U.S. Patent Publication No. 2011/0171737, incorporated by reference) uses stochastic optimization algorithms that choose different codons within an ORF without altering the amino acid sequence to optimize a target function designed to identify mRNA sequences that express proteins at high levels in E. coli. The OrfOpt simultaneously imposes AU-rich nucleotide composition at the 5′ and 3′ ends of the ORF, low RNA secondary structure content and favorable codon usage (Allert et al., 2010, J Mol Biol, 402, 905-18). The OrfMorph program reproduces the pattern of codon usage and RNA secondary structure observed in the parent genome of a protein, but using E. coli codon preferences and nucleotide composition.
[0512] Codon usage is calculated using the codon adaptation index (CAI), as described for OrfOpt, using codon frequency tables calculated for the genome under examination. The mean CAI value for a genome, μ.sub.c, and its standard deviation, σ.sub.c, are calculated over all the codons in a genome. A codon usage score, c, is calculated for each codon in an open reading frame (ORF) by averaging the CAI over a 9-codon window, centered on the codon for which this score is calculated. A normalized codon usage score, z.sub.c, is calculated for each codon as Z-score: z.sub.c=(c−μ.sub.c)/σ.sub.c. A plot of z.sub.c along an ORF establishes the codon usage pattern of that ORF. Rare codons (z.sub.c<0) are hypothesized to slow down the elongation rate of ribosome translation, introducing “pause” sites at extreme values. Such pause sites are hypothesized to direct kinetics of co-translational folding, allowing a newly synthesized segment to fold before more protein is made. An RNA secondary structure score, s, is determined for each nucleotide by summing its participation in all possible hairpins that can form in its vicinity (settings: minimum duplex length 4 basepairs; maximum loop length, 30 bases; vicinity length, 100 bases), as described for OrfOpt. The average secondary structure energy, μ.sub.s, and its standard deviations, σ.sub.s, are calculated over all the nucleotides in a genome. A normalized secondary structure energy score, z.sub.s, is calculated for codon as the Z-score: z.sub.s=(c−μ.sub.s)/σ.sub.s. A plot of z.sub.s along an ORF establishes the secondary structure pattern of that ORF. Regions of above-average secondary structure (z.sub.s>0) are hypothesized to slow down the elongation rate of ribose translation, introducing “pause” sites at extremes. As with CAI-mediated pause sites, secondary structure-driven pause sites are hypothesized to direct the kinetics of co-translational folding.
[0513] To mimic these patterns for heterologous expression of an ORF in E. coli, first the z.sub.c and z.sub.s scores are calculated using the parent organism codon table, μ.sub.c, σ.sub.c, μ.sub.s, and σ.sub.s values. Second, a stochastic search algorithm is used that randomly chooses between degenerate codons to construct trial mRNA nucleotide sequences, calculating z.sub.c and z.sub.s scores for each trial sequence, but using the E. coli codon table, and E. coli μ.sub.c, σ.sub.c, μ.sub.s, and σ.sub.s values. For each trial, the absolute differences between the E. coli trial scores, and the wild-type scores are summed over the entire ORF. The OrfMorph program searches for a minimum of these differences. The stochastic search algorithm operates by first choosing a codon position, second choosing a degenerate codon within the allowed codons at that position. If the choice results in an improved score, the sequence is kept, otherwise it is rejected. After a position has been selected, it is removed from the pool of allowed positions, and the next is chosen from the remainder. The algorithm terminates when two successive sweeps do not yield further improvements in the score. The resulting RNA nucleotide sequence that has codon usage patterns and secondary structure patterns that closely match those of the wild-type mRNA sequence in its parental genomic context. The strategy is that such matching improves production of soluble protein by mimicking co-translational folding contributions that minimize mis-folded protein intermediate aggregation.
[0514] Protein expression, purification, and fluorescent conjugate preparation. Plasmids carrying the expression constructs (see above) were transformed into KRX competent cells (Promega), and grown overnight at 37° C. on LB agar plates (100 mg/mL ampicillin). A single colony was picked and grown overnight at 37° C. in Terrific Broth (TB; Research Products International). The overnight cultures were diluted 1:20 in 500 mL TB (100 mg/mL ampicillin), grown to an optical density of A.sub.600=0.5 at 37° C. in vigorously aerated shaker flasks, induced by the addition of 2.5 mL rhamnose (20% w/v), and grown for a further 3-4 hrs. The cells were harvested by centrifugation (5,000 rpm, 10 min). After decanting the supernatant, the cell pellets were stored −80° C. The cell pellets were thawed, resuspended in 8 mL binding buffer (10 mM imadozole, 20 mM MOPS, 500 mM NaCl, pH 7.8). Following resuspension, 3 mL of BugBuster HT (EMD Millipore) was added. After incubation (20 mins, 25° C.), the cells were lysed on ice by sonication (2 minutes of one-second on/off pulses, 20-30% power). A clarified lysate was prepared by centrifugation (15,000 rpm, 20 min, 4° C.) from which recombinant protein was purified by batch immobilized metal affinity chromatography (IMAC). Resuspended IMAC agarose beads (5 mL; Sigma-Aldrich, P6611) were added to the lysate. After incubation at 4° C. in a Mini LabRoller (Labnet International) for 1 hr, the beads were washed at least five times with binding buffer. The immobilized protein beads were resuspended in labeling buffer (20 mM MOPS, 100 mM NaCl, pH 6.9) and labeled overnight (4° C., rotating end-over-end) with a thiol-reactive fluorophore (5-fold stoichiometric excess over protein). Following two rinses with labeling buffer to remove unincorporated label, the proteins were eluted from the beads. For double labeling of βZif fusions, a second thiol-reactive label was added following reduction of the disulfide with 5 mM TCEP. To elute labeled protein from the IMAC beads, 6 mL of elution buffer (400 mM imidazole, 500 mM NaCl, 20 mM MOPS, pH 7.8) was added, incubated for 30 min (4° C., rotating end-over-end), and the beads removed by centrifugation. Following dialysis of the eluate against three changes of assay buffer (20 mM MOPS, 20 mM KCl, pH 7.4), using 10 kDa semi-perimeable membrane (Snakeskin tubing, Thermo Scientific), the fluorescent conjugates were concentrated in a 10 kDa cutoff spin concentrator (Vivaspin, GE Healthcare). Protein purity was assessed by SDS/PAGE. Protein concentrations were determined by (Nanodrop1000) at 280 nm (using extinction coefficients calculated from their sequence (Gill and von Hippel, 1989, Anal Biochem, 182, 319-26; Artimo et al. 2012, Nucleic Acids Res, 40, W597-603), or at the fluorophore absorbance peak (Acrylodan, 391 nm and Badan, 387 nm).
[0515] Determination of temperature- and ligand-dependent fluorescence landscapes. 12-, 24-, or 48-point logarithmic titration series were prepared on a Tecan Freedom liquid-handling robot, using an in-house program, ‘TitrationPlate’, that compiles an abstract description of a multi-component titration series into machine instructions for operating the robot. Temperature-dependent fluorescence emission intensities of 20 μL aliquots, each containing 10 μM protein, were measured in 384-well microtiter plates in a LightCycler 480 II (Roche) using excitation and emission wavelengths available for this instrument that most closely matched the optical characteristics of the fluorescent conjugate. Temperatures were advanced in 1K. steps. At each temperature, data was collected at 1-second intervals for 60 seconds at which point the signal had relaxed to a steady value associated with the new temperature. Under these experimental photobleaching was not observed. The in-house program ‘TitrationMeltPlate’ was used to convert these observations into time-independent datasets that record fluorescence as a function of temperature for each well and associate wells with their concentration of titrant and additive. Management tools were developed to maintain a database of titrations and their analyses.
[0516] Determination of emission intensity spectra. Ligand- and wavelength-dependent emission intensities were recorded on a Nanodrop3300 (Thermo Scientific) at room temperature. Using the LED closest to the optimal excitation wavelength of the fluorophore (UV, 365 nm; blue, 470 nm; ‘white’, 460-550 nm).
[0517] Ratiometric analysis of glucose binding. Isothermal glucose titrations were extracted from the fluorescent landscape or emission spectra datasets obtained as described above. Monochromatic emission intensities I.sub.λ (these intensities correspond to a bandpass intensity, recorded either with a physical filter in the case of the Roche LightCycler, or by integrating in the interval λ−δ, λ+δ in the case of an emission spectrum), were fit to
I.sub.λ=.sup.apoβ.sub.λ(1−
where .sup.apoβ.sub.λ and .sup.satβ.sub.λ are the fluorescence baselines associated with the ligand-free and ligand-bound states of the protein, respectively, and
.sup.satβ.sub.x=a.sub.x+b.sub.x[L] 2
For a single glucose-binding site, the fractional saturation is given by
where [L] is the ligand (glucose) concentration and K.sub.d the dissociation constant, .sup.trueK.sub.d for
[0518] A ratiometric signal at a given point in a titration series, R.sub.12(t), is given by the ratio of intensities at two wavelengths, .sup.obsI(λ.sub.1,t), .sup.obsI(λ.sub.2,t) in the emission spectrum measured at that point:
where a.sub.t is an attenuation factor that describes the effect of variations in sample size (i.e. the amount of observable fluorophore) in the t.sup.th sample on the wavelength-independent intensity of the entire emission spectrum. This signal removes wavelength-independent emission intensity attenuation effects due to variations in conjugate concentration, photobleaching, fluctuations in excitation source intensities, and detection efficiency (Demchenko, 2010, J Fluoresc, 20, 1099-128; Demchenko, 2014, Journal of Molecular Structure, 1077, 51-67). It is a key aspect for high-precision sensing using the reagentless fluorescently-responsive sensors described here. The ratiometric signal also can be fit to a binding isotherm:
R.sub.1,2=.sup.apoβ.sub.R(1−
where .sup.apoβ.sub.R and .sup.satβ.sub.R are the baselines, and
where .sup.apoI.sub.λ2 and .sup.satI.sub.λ2 are the emission intensities of the monochromatic signal at wavelength λ.sub.2 of the ligand-free and ligand-bound protein, respectively.
[0519] Following a fit of the titration series using equations 4 and 5, a.sub.t values can be recovered by taking the average comparison of the observed and calculated intensities at the two wavelengths:
The a.sub.t value can then be applied to all wavelengths to obtain an emission spectrum or integrated intensity of the t.sup.th titration point corrected for variations in sample size:
.sup.corrI(λ)=a.sub.t.sup.obsI(λ) 8
where .sup.corrI(λ) and .sup.obsI(λ) are the wavelength-dependent intensities of the corrected and observed emission spectra, respectively.
[0520] The fractional error in the chemometric concentration measurement, depends on the first derivative of the binding isotherm as follows (Marvin et al., 1997, Proc Natl Acad Sci USA, 94, 4366-71):
Where R.sub.1,2 is the ratiometric signal (equation 5), ϵ.sub.1,2 its experimental error, and δS is the resulting chemometric error in the concentration. We can then define a relative precision function
where P(S) is the relative precision at concentration S, which reaches a maximum value (i.e. lowest error), P.sub.max, at the K.sub.d.
[0521] For a given isothermal titration, values for .sup.appK.sub.d and .sup.trueK.sub.d were obtained using a non-linear fitting algorithm in which these two parameters were simultaneously fit to the three experimental binding isotherms using equations 1 and 5, with the two monochromatic isotherms sharing the same .sup.trueK.sub.d value. Three separate pairs of .sup.apoβ and .sup.satβ were fit in this procedure, corresponding to the two monochromatic and the ratiometric signals, respectively. Two distinct ratiometric response models can be used: coupled (both wavelengths respond to ligand); uncoupled (the second wavelength is non-responsive; i.e. remains constant). Optionally, an attenuation vector, a(t) containing a.sub.t values for each titration point (equation 7), can be refined by iterative fit cycles in which the a(t) vector of a previous cycle is used to adjust the integrated intensities of the next cycle. Programs ‘Nanodrop3300’ and ‘TitrationMeltAnalysis’ were developed to analyze wavelength- or temperature-dependent ligand-binding datasets respectively.
[0522] Analysis of glucose-binding properties using thermal melts. The thermal stability of purified GBP candidate proteins was determined by measuring the temperature-dependence of the fluorescence signal of an extrinsically added dye, SYPRO, using a Roche LightCycler (Layton and Hellinga, 2010, Biochemistry, 49, 10831-41). The total fluorescence intensity, S, is given by
S=β.sub.Ff.sub.F+β.sub.Uf.sub.U 11
where f.sub.F and f.sub.U are the fractions of protein in the folded and unfolded states, respectively, and β.sub.F and β.sub.U the fluorescence baselines of these two states. To get the fractions of the two states, we have
where K.sub.U(T) is the temperature-dependent unfolding equilibrium constant, which by the van't Hoff approximation is given by
Where T is the temperature, T.sub.m, the unfolding reaction transition mid-point temperature, and ΔH.sub.U the enthalpy of unfolding.
[0523] To obtain the temperature dependence of the binding reaction, the K.sub.d values of all the individually determined isotherms were fit the Gibbs-Hemholtz equation (Layton and Hellinga, 2010, Biochemistry, 49, 10831-41):
where ΔG.sub.b*(T) is the standard free energy of binding at 1 M ligand at temperature T,
Δ.sup.refH.sub.b* and Δ.sup.refS.sub.b* the molar enthalpy and entropy of binding, respectively, at the reference temperature, T.sub.ref, and ΔC.sub.p,b the heat capacity of the binding reaction. This data analysis was carried out using ‘TitrationMeltAnalysis’.
[0524] Mechanisms for Ligand Sensing Using Non-Geometric Modulation of FRET.
[0525] The subject matter disclosed herein is not limited to or bound by any particular scientific theory. However, discussions regarding ngmFRET are provided to facilitate the understanding of possible mechanisms involved with ngmFRET signaling in various embodiments described herein. Equations for calculating various values mentioned herein are also provided.
[0526] The total signal, S, of a fluorescent sensor (either single-wavelength emission intensities, I.sub.λ, or ratios of intensities at two wavelengths, R.sub.12) is the sum of the fluorescence due to the ligand-free (apo) and ligand-bound states:
S=α(1−
where α and β are the fluorescent baselines in the ligand-free and -bound states, respectively, and
[0527] Fluorescence quantum yields are the fractions of photons emitted by the excited state relative to the total absorbed, and correspond to the ratio of the radiative decay rate relative to the sum of the rates of all possible decay pathways (
where k.sub.r and k.sub.nr are the radiative and non-radiative decay rates of the excited state, respectively. If we define q as the ratio between the radiative and non-radiative decay rates,
then the quantum yield can be written as
[0528] Chemical sensors exploit the ligand-mediated shift of a fluorescent system between the ligand-free and ligand-bound states which each exhibit distinct quantum yields:
Q.sub.obs=Q.sub.apo(1−
where Q.sub.obs, Q.sub.apo and Q.sub.sat are the quantum yield of the total system, the apo-protein, and the ligand-bound complex, respectively. In a system involving energy transfer between a donor and acceptor fluorophore, the Q.sub.apo and Q.sub.sat quantum yields each are combinations of their respective donor and acceptor quantum yields:
Q.sub.apo=.sup.DQ.sub.apo+.sup.AQ.sub.apo and Q.sub.sat=.sup.DQ.sub.sat+.sup.AQ.sub.sat 21
where the superscripts D and A indicate donor and acceptor fluorophores respectively. To understand ngmFRET-based sensors, we therefore need to examine the factors that affect each of these four quantum yields.
[0529] The intensity of the light emitted by a donor or its acceptor is determined by the rate of photon emission from their respective excited states (
[0530] The rate of resonance energy transfer, k.sub.t, along a non-radiative pathway between donor and acceptor (
k.sub.t=φQ.sub.D.sup.Dk.sub.r 22
where Q.sub.D is the donor quantum yield in the absence of an acceptor.
[0531] According to the Förster model of weakly coupled oscillators (Lakowicz, 2006, Principles of fluorescence spectroscopy. Springer, New York; Valeur, 2012, Molecular Fluorescence. Principles and Applications. Weinheim: Wiley), the energy transfer coupling factor is dependent on the spectral overlap, J, of the donor emission, .sup.Dλ.sub.em, and acceptor excitation spectrum, .sup.Aλ.sub.ex, and the variation of the geometry, G, between the donor and acceptor excited state transition dipoles with distance, r, and orientation factor, κ:
and
J(.sup.Dλ.sub.em,.sup.Aλ.sub.ex)=∫F(.sup.Dλ.sub.em)ε(.sup.Aλ.sub.ex)λ.sup.Adλ 25
with n the refractive index of medium, N.sub.A Avogrado's number, F(.sup.Dλ.sub.em) the normalized donor emission spectrum, and ε(.sup.Aλ.sub.ex) the absorption coefficient of the acceptor excitation spectrum [this analysis is a re-arrangement of the traditional presentation of the equations describing tgmFRET, separating the different contributions (geometry, spectral overlap, quenching)]. Ligand-mediated modulation of r, κ and J therefore affects k.sub.t (
[0532] At steady state, the concentration of the donor excited state, [D*], is given by the following rate balance equation (see
N.sub.0αk.sub.ex−[D*](.sup.Dk.sub.nr+.sup.Dk.sub.r+k.sub.t)=0 26
where N.sub.0 is the population of ground state fluorophores, k.sub.ex the rate of excitation photon absorption, α the effective illumination, k.sub.t, the resonance energy transfer rate, .sup.Dk.sub.nr and .sup.Dk.sub.r the radiative and non-radiative decay rates of the donor (by itself and regardless of the presence of any other fluorophore/parter) in the absence of acceptor, respectively. Substituting .sup.Dk.sub.r(d+1) for .sup.Dk.sub.r+.sup.Dk.sub.nr, (using equation 18, with d=q, the ratio of non-radiative to radiative decay rates in the donor), and replacing k.sub.t with equation 22 (with Q.sub.D=1/(1+d), according to equation 23), we obtain
The intensity of the emitted donor light, I.sub.D, is
The donor quantum yield, Q.sub.D, is this emission intensity relative to the intensity of the excitation, k.sub.exαN.sub.0
[0533] The rate balance equation for the acceptor excited state concentration, [A*], is given by
[D*]k.sub.t−[A*](.sup.Ak.sub.r+.sup.Ak.sub.nr) 31
Consequently, by applying equations 19, 22 and 30, the acceptor quantum yield, Q.sub.A, is
where a is the ratio of the radiative and non-radiative pathways in the acceptor.
[0534] The ratio of the acceptor and donor quantum yields therefore is
This equation clearly shows that any ligand-mediated change in energy transfer (ϕ) or quenching of either the donor (d) or acceptor (a) leads to a change in the ratio of donor and acceptor emission intensities, thereby enabling ratiometry.
[0535] Classical ligand-mediated modulation of tgmFRET is concerned only with ligand-mediated changes in the distance between the donor and acceptor (Clegg, 1995, Curr. Opin. Biotechnol., 6, 103-110; Cheung, 1991, Topics in Fluorescence Spectroscopy, 2, 127-176), and does not take advantage of effects that alter the photophysics of individual chromophores. By contrast, in ngmFRET systems, the directly responsive partner (DRP) responds to ligand binding through ligand mediated changes that alter the ratio of its radiative and non-radiative pathways (quenching, d or a) or its spectral properties (J), whereas the indirectly responsive partner (IRP) changes only as a consequence of the effect that such change have on the resonance energy transfer rate (k.sub.t). It is important to realize that the DRP can function either as a ngmFRET donor an acceptor, depending on how the spectral overlap is set up with the IRP. Regardless of whether the DRP is a donor or acceptor, ligand-mediated alteration of its non-radiative to radiative decay rate ratio (parameter d for a DRP donor; a for an acceptor; by itself and regardless of the presence of any other fluorophore/parter) changes its emission intensity. In DRP donors quenching also alters the ngmFRET transfer rate (see equations 22 and 27), thereby changing the emission intensities of not only itself but also its IRP. By contrast, in DPR acceptors quenching does not alter ngmFRET, and hence do not affect its IRP donor intensity. A DRP acceptor therefore can alter intensities of its donor IRP only if ligand binding changes ϕ. If the DRP is a donor, then manipulation of the ngmFRET coupling factor, ϕ, changes the rate of excited state decay; if it is an acceptor, the rate of excited state formation is altered.
[0536] Regardless of whether the DRP is a donor or acceptor, a change in any of the two parameters (ϕ and d or a) alters the ratio of the donor and acceptor quantum yields (equation 33), thereby enabling ratiometry. Ligand-mediated donor DRP quenching affects the quantum yields of both the donor, Q.sub.D, and acceptor, Q.sub.A, quantum yields (equations 30, 32). Quenching of an acceptor DRP alters only Q.sub.A (equation 30). Changes in ϕ affect quantum yields of both fluorophores, regardless whether the DRP functions as the donor or acceptor (equations 23-25, 30, 32). For systems in which there is no ligand-mediated change in the (average) distance between the two fluorophores, ϕ changes only if the DRP switches between two different excited state populations (“dipole switching”) in response to ligand binding and if the two excited states differ in their spectral properties (emission for donor DRPs; absorption for acceptor DRPs). Excited state dipoles usually also differ in their dipole orientations, so it is likely that changes in spectral overlap involve (re-)orientation effects. They are also likely to differ in the relative rates of their radiative and non-radiative decay rates. Dipole switching therefore is likely to involve a combination of changes in ngmFRET and quenching effects.
[0537] There are eight possible combinations of ligand-mediated changes in quenching and ngmFRET parameters, which have different outcomes on the two emission intensities and their ratio, depending on whether the DRP is the donor or acceptor. The qualitative behavior of the resulting sixteen possibilities in ngmFRET systems are shown in Table 7. Twelve of these have a predictable outcome on the direction of change in the ratio of the two emission intensities. The effect on the direction of change for both donor and acceptor emission intensities can be predicted for seven models. For the other models, the direction of change of one or both peaks depends on the size of the change in the underlying parameters. Purely geometric effects (changes in inter-dipole distance or orientation) always result in anti-correlated changes in emission intensity changes (i.e. one increases and the other decreases, or vice versa). Correlated (i.e both intensities increase or decrease) or uncorrelated (one changes, the other remains constant) intensity changes therefore are prima facie evidence for an ngmFRET effect.
Example 9. Glucose Biosensors and Uses Thereof
[0538] We report the construction of a robust, thermostable, reagentless, fluorescently responsive glucose biosensor and its variants derived from Thermus scotoductus (tsGBP2). These engineered proteins can be used for high-precision chemometric measurements that span the entire clinical glucose concentration range, using fluorescence ratiometry measured with straightforward, inexpensive instrumentation.
[0539] Thermostable homologs of the Thermus thermophilus glucose-galactose binding protein (ttGBP1) were identified using a bioinformatics search strategy that applied a structure-based sequence filter to identify the subset of sequences that retain the original function within the larger collection of aligned sequence homologs. The homologs tested appeared at sequence identities from 32% to 91% of the ttGBP1 probe. At levels below 60%, overall identities are weak predictors of biological function (Todd, 2001, J. Mol. Biol., 307, 1113-1143; Tian, 2003, J. Mol. Biol., 333, 863-882; George, 2005, Proc Natl Acad Sci USA, 102, 12299-12304), application of the structure-based filter therefore was essential for accurate identification. The glucose-binding properties of the predicted hits were tested experimentally by constructing synthetic genes optimized for heterologous protein expression in E. coli (Allert, Cox and Hellinga, 2010, J Mol Biol, 402, 905-18) and determining the glucose-binding properties of the expressed proteins. This search resulted in the identification of a homolog from Thermus scotoductus (tsGBP2) as a suitable candidate for glucose sensor engineering.
[0540] Endosterically placed Acrylodan fluorescent conjugates were found to be highly effective ratiometric glucose sensors. The strongest dichromatic response was observed for the 13C⋅Acrylodan conjugate. We demonstrated that the signaling properties of conjugates attached to this position can be conserved throughout the family of ttGBP1 homologs. We also showed that signaling properties can be manipulated using site-specific double labeling to set up ngmFRET systems in which one partner is directly responsive to glucose binding.
[0541] A series of additional mutations were introduced to manipulate glucose affinities. Variants spanning four orders of magnitude (0.1-100 mM) were identified. Within these, a subset of mutants covers the entire pathophysiological glucose concentration range with responses that remain within 90% of the maximally achievable precision.
[0542] The tsGBP2-based FRSs can be immobilized site-specifically on magnetic beads without affecting protein stability or fluorescence responses. They can be dried, and aged aggressively (incubation at 50° C. for 7 days) without adversely affecting sensor performance. These results demonstrate some of the advantages of using hyperthermostable proteins.
[0543] Reagentless, fluorescently responsive sensors present a number of advantages over enzyme-based biosensors, including self-calibration, elimination of chemical transformations and multiple substrates, which together lead to simple sample-handling fluidic circuitry and rapid response times. FRSs can be used for one-time, episodic, and continuous monitoring measurements. Additionally, the use of robust engineered glucose sensors based on (hyper) thermophilic proteins is likely to simplify manufacturing and distribution processes. Combinations of mutant glucose sensors reported here into multiplexed arrays or composites can determine glucose concentrations from hypoglycemic to the hyperosmolar hyperglycemic state samples with high precision in one measurement. Such systems have significant potential for the development of next-generation high-accuracy, wide dynamic range sensing applications in continuous monitoring, point-of-care, or wearable systems.
[0544] The glucose sensors can be incorporated into point-of-care clinical devices to measure glucose concentrations accurately, and rapidly at the patient bedside. In such a device, a small blood sample (<10 μL) is obtained by means of a finger stick using a lancet. This sample droplet is then placed on the aperture of a disposable cartridge containing desiccated, immobilized glucose sensors inside a small measurement chamber. The sample enters the chamber by virtue of passive capillary action, wetting the sensors upon contact. As soon as the sensors have been wetted, they bind glucose, and report on its concentration by virtue of the engineered fluorescent sensor mechanism. The cartridge is placed inside a small reader (handheld or on a desktop), and their fluorescence signal is measured by the (inexpensive) optoelectronic components of the reader. Excitation light is provided by a light-emitting diode (LED). In the case of Acrylodan or Badan, a commercially available 400 nm blue LED is used, and the emitted light is measured through two bandpass filters. Cartridges can contain multiple sensors, spanning the entire clinical range of possible glucose concentrations. Each sensor is immobilized at a particular, known location inside the cartridge, providing “spatial addressability”. The intensity at a particular wavelength is then recorded by imagining these sensors using an inexpensive camera, such as a Complementary metal-oxide semiconductor (CMOS) device commonly found in consumer electronics such as cell phones. Each pixel in the camera records the emitted light on a gray scale. Integration of that signal imaged through the two signals, is analyzed by an on-board computer to calculate the ratiometric signal for each immobilized sensor. Pre-recorded hyperbolic binding curves are then used to calculate the glucose concentration in the sample. Recording through multiple sensors, tuned for accurate detection at different glucose concentrations provides a high-accuracy reading. This process is completed in less than a minute.
[0545] Similar instrumentation can be used for any type of episodic measurements, for instance, using other bodily fluids, or samples obtained from animals, or non-biological samples such as foods and beverages.
[0546] The FRS glucose sensors also can be used to monitor glucose levels continuously. For instance, sensors can be immobilized at the tip of a thin optical fiber to construct a glucose-responsive optode. Such an optode can be introduced into the body subcutaneously, using a small needle. Excitation and emission light are passed to and from the immobilized sensor, respectively. The sensor is in continuous contact with the sample. Fluctuations in the glucose sample alter the dynamic equilibrium between the open and closed states of the glucose-binding protein, which is transduced into fluctuations of the fluorescent emission signal, by virtue of the sensing mechanism of the conjugated fluorophore. The emitted light intensities are read through filters by a reader connected to the optode. This reader continuously displays the change in signal, and the corresponding calculated glucose concentrations. Continuous glucose monitoring accomplished using a device containing the immobilized glucose biosensor(s), e.g., a fiber optic biosensor, introduced into the subject intradermally or subcutaneously (Judge et al., 2011, Diabetes Technology & Therapeutics 13 (3):309-317; Weidemaier et al., 2011, Biosensors and Bioelectronics 26:4117-4123; hereby incorporated by reference).
[0547] As was discussed above, the features that distinguish the described constructs, devices, and methods from earlier glucose assay systems include: [0548] Self-calibration [0549] Rapid response time [0550] Simple sample-handling fluidic circuitry [0551] No additional components/substrates (“reagentless”) [0552] No incubation time to develop signal. Reading is near-instantaneous and continuous [0553] Stability (simplifies manufacturing, distribution, storage) [0554] Small sample volume (<10 μL). [0555] Capable of precise measurements over extended glucose concentration range (from the hypoglycemic to the hyperglycemic-hyperosmotic range) [0556] Multiple sensors also provides redundancy, lowering error [0557] Large scope of uses: episodic, continuous, ex vivo, in vivo, optodes, implants, dermal patches.
OTHER EMBODIMENTS
[0558] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
[0559] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[0560] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.