Thermostable Proteins and Methods of Making and Using Thereof

Abstract

The present invention relates to functional, modified glucose-galactose binding proteins (GGBPs), that have a greater melting temperature (T m ) than a reference GGBP. The present invention also relates to biological sensors, e.g., glucose sensors, comprising these thermostable GGBPs. The present invention also relates to nucleic acids encoding these thermostable GGBPs.

1 . A modified thermostable glucose-galactose binding protein (GGBP) that has a higher melting temperature than a reference GGBP. 
     
     
         2 . The modified thermostable GGBP of  claim 1 , wherein said reference GGBP is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. 
     
     
         3 . (canceled) 
     
     
         4 . The modified thermostable GGBP of  claim 2 , wherein said modified thermostable GGBP comprises one or more residue substitutions selected from the group consisting of residues corresponding to tyrosine 12 (Y12), asparagine 15 (N15), asparagine 39 (N39), serine 41 (S41), asparagine (N43), asparagine 49 (N49), aspartic acid 50 (D50), glutamine 51 (Q51), isoleucine (152), glycine 82 (G82), glutamine 83 (Q83), asparagine 84 (N84), alanine 128 (A128), alanine 129 (A129), asparagine 130 (N130), glutamine (Q131), glycine 132 (G132), tryptophan 133 (W133), glutamine 175 (Q175), glutamine 177 (Q177), leucine 178 (L178), glycine 198 (G198), proline 199 (P199), asparagine 200 (N200), asparagine (N202), lysine (K203) asparagine 226 (N226), asparagine 259 (N259), asparagine 260 (N260), aspartic acid 267 (D267), leucine, 268 (L268), alanine 269 (A269), lysine 270 (K270), asparagine 271 (N271), asparagine 283 (N283), tryptophan 284 (W284) and asparagine 302 (N302) of SEQ ID NO:1. 
     
     
         5 . (canceled) 
     
     
         6 . The modified thermostable GGBP of  claim 2 , wherein said modified thermostable GGBP comprises one or more residue substitutions selected from the group consisting of residues corresponding to tyrosine 35 (Y35), asparagine 38 (N38), asparagine 62 (N62), serine 64 (S64), asparagine 66 (N66), asparagine 72 (N72), aspartic acid 73 (D73), glutamine 74 (Q74), isoleucine 75 (175), glycine 105 (G105), glutamine 106 (Q106), asparagine 107 (N107), alanine 151 (A151), alanine 152 (A152), asparagine 153 (N153), glutamine 154 (Q154), glycine 155 (G155), tryptophan 156 (W156), glutamine 198 (Q198), glutamine 200 (Q200), leucine 201 (L201), glycine 221 (G221), proline 222 (P222), asparagine 223 (N223), asparagine 202 (N225), lysine 226 (K226) asparagine 249 (N249), asparagine 282 (N282), asparagine 283 (N283), aspartic acid 290 (D290), leucine 291 (L291), alanine 292 (A292), lysine 293 (K293), asparagine 294 (N294), asparagine 306 (N306), tryptophan 307 (W307) and asparagine 325 (N302) of SEQ ID NO:2. 
     
     
         7 - 10 . (canceled) 
     
     
         11 . The modified thermostable GGBP of  claim 2 , wherein said modified thermostable GGBP comprises one or more residue substitutions selected from the group consisting of residues corresponding to tyrosine 12 (Y12), asparagine 15 (N15), asparagine 39 (N39), serine 41 (S41), asparagine (N43), asparagine 49 (N49), aspartic acid 50 (D50), glutamine 51 (Q51), isoleucine (152), glycine 82 (G82), glutamine 83 (Q83), asparagine 84 (N84), alanine 128 (A128), alanine 129 (A129), asparagine 130 (N130), glutamine (Q131), glycine 132 (G132), tryptophan 133 (W133), glutamine 175 (Q175), glutamine 177 (Q177), leucine 178 (L178), glycine 198 (G198), proline 199 (P199), asparagine 200 (N200), asparagine (N202), lysine (K203) asparagine 226 (N226), asparagine 259 (N259), asparagine 260 (N260), aspartic acid 267 (D267), leucine 268 (L268), alanine 269 (A269), lysine 270 (K270), asparagine 271 (N271), asparagine 283 (N283), tryptophan 284 (W284) and asparagine 302 (N302) of SEQ ID NO:4. 
     
     
         12 . The modified thermostable GGBP of  claim 11 , wherein said modified thermostable GGBP comprises the substitutions N391, G82E, 083K, N84D, Q175E, Q177H, L178M, N259E and N260S. 
     
     
         13 . A biosensor for measuring an analyte, said biosensor comprising the modified thermostable GGBP of  claim 11 . 
     
     
         14 . The biosensor of  claim 13 , wherein said modified thermostable GGBP further comprises at least one label selected from the group consisting of a fluorescent label, a radio label, a chemiluminescent label, a phosphorescent label, a colorimetric label and a surface-enhanced raman-scattering label. 
     
     
         15 . The biosensor of  claim 13 , wherein said thermostable GGBP is entrapped in a polymeric matrix. 
     
     
         16 . A method of measuring the concentration of an analyte in a sample, said method comprising contacting the biosensor of  claim 15  with said sample. 
     
     
         17 . A device comprising the biosensor of  claim 13 , wherein said thermostable GGBP is in optical proximity with an optical conduit. 
     
     
         18 . A biosensor for measuring an analyte, said biosensor comprising the modified thermostable GGBP of  claim 4 . 
     
     
         19 . The biosensor of  claim 18 , wherein said modified thermostable GGBP further comprises at least one label selected from the group consisting of a fluorescent label, a radio label, a chemiluminescent label, a phosphorescent label, a colorimetric label and a surface-enhanced raman-scattering label. 
     
     
         20 . The biosensor of  claim 18 , wherein said thermostable GGBP is entrapped in a polymeric matrix. 
     
     
         21 . A method of measuring the concentration of an analyte in a sample, said method comprising contacting the biosensor of  claim 20  with said sample. 
     
     
         22 . A device comprising the biosensor of  claim 18 , wherein said thermostable GGBP is in optical proximity with an optical conduit. 
     
     
         23 - 32 . (canceled) 
     
     
         33 . An isolated nucleic acid encoding the modified thermostable GGBP of  claim 1 . 
     
     
         34 . A vector comprising the nucleic acid of  claim 33 . 
     
     
         35 . A host cell comprising the vector of  claim 34 . 
     
     
         36 . A method of making a modified thermostable GGBP, said method comprising culturing the host cell of  claim 35  in conditions suitable for protein expression and isolating said protein.