Thermostable glucose biosensors and uses thereof

Abstract

The present subject matter provides glucose biosensors as well as compositions, devices, and methods comprising such biosensors.

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 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)