Binding proteins as biosensors

Abstract

The invention is directed to compositions of mutated binding proteins containing reporter groups, analyte biosensor devices derived therefrom, and their use as analyte biosensors both in vitro and in vivo.

1 . A method for glucose detection comprising: 
 a) providing at least one mutated glucose/galactose binding protein and at least one reporter group attached thereto;    b) exposing said mutated glucose/galactose binding protein to varying glucose concentrations; and    c) detecting a detectable and reversible signal change from said reporter group wherein said detectable and reversible signal change corresponds to said varying glucose concentrations.    
   
   
       2 . The method of  claim 1  wherein said detecting is continuous, programmed, episodic, or combinations thereof.  
   
   
       3 . The method of  claim 1  wherein said mutated glucose/galactose binding protein is selected from bacterial periplasmic binding proteins.  
   
   
       4 . The method of  claim 1  wherein said detecting of detectable and reversible signal changes from said reporter group of varying glucose concentrations is in vivo.  
   
   
       5 . The method of  claim 1  wherein said binding protein comprises one amino acid substitution.  
   
   
       6 . The method of  claim 5  wherein said binding protein comprises at least two amino acid substitutions.  
   
   
       7 . The method of  claim 6  wherein said binding protein comprises at least three amino acid substitutions.  
   
   
       8 . The method of  claim 5  wherein said one amino acid substitution is selected from the group consisting of a cysteine at position 1, a serine at position 1, a cysteine at position 11, a cysteine at position 14, a cysteine at position 19, a cysteine at position 43, a cysteine at position 74, a cysteine at position 107, a cysteine at position 110, a cysteine at position 112, a cysteine at position 113, a cysteine at position 137, a cysteine at position 149, a cysteine at position 213, a cysteine at position 216, a cysteine at position 238, a cysteine at position 287, a cysteine at position 292, a cysteine at position 152, a cysteine at position 182, a cysteine at position 236, and a cysteine at position 296.  
   
   
       9 . The method of  claim 8  wherein said mutated glucose/galactose binding protein additionally comprises at least one histidine tag.  
   
   
       10 . The method of  claim 6  wherein said mutated glucose/galactose binding protein has at least two amino acid substitutions selected from the group consisting of a cysteine at position 112 and a serine at position 238, a cysteine at position 149 and a serine at position 238, a cysteine at position 152 and a serine at position 213, a cysteine at position 213 and a cysteine at position 238, a cysteine at position 149 and an arginine at position 213, a cysteine at position 149 and a cysteine at position 213, a cysteine at position 149 and a threonine at position 213, a cysteine at position 149 and a leucine at position 213, a cysteine at position 149 and a tyrosine at position 213, a cysteine at position 149 and a serine at position 213, a cysteine at position 149 and an asparagine at position 223, a cysteine at position 149 and a cysteine at position 238, a cysteine at position 149 and a serine at position 256, a cysteine at position 149 and an arginine at position 256, a cysteine at position 152 and an arginine at position 213, a cysteine at position 152 and an asparagine at position 223, a cysteine at position 213 and a cysteine at position 255.  
   
   
       11 . The method of  claim 10  wherein said mutated glucose/galactose binding protein additionally comprises at least one histidine tag.  
   
   
       12 . The method of  claim 7  wherein said glucose/galactose binding protein has at least three amino acid substitutions selected from the group consisting of a cysteine at position 149, a serine at position 213 and a serine at position 238; a cysteine at position 149, an arginine at position 213 and a serine at position 238; a cysteine at position 149, a cysteine at position 213 and a cysteine at position 238; a cysteine at position 149, a serine at position 213 and an asparagine at position 223; a cysteine at position 149, an asparagine at position 223 and an arginine at position 256; a cysteine at position 149, an arginine at position 213 and a cysteine at position 238; and a cysteine at position 149, a cysteine at position 213 and a serine at position 238.  
   
   
       13 . The method of  claim 12  wherein said mutated glucose/galactose binding protein additionally comprises at least one histidine tag.  
   
   
       14 . The method of  claim 6  wherein said binding protein comprises at least four amino acid substitutions.  
   
   
       15 . The method of  claim 14  wherein said four amino acid substitutions are selected from the group consisting of a serine at position 1, a cysteine at position 149, an arginine at position 213 and a serine at position 238; and a cysteine at position 149, a cysteine at position 182, a cysteine at position 213 and a serine at position 238.  
   
   
       16 . The method of  claim 15  wherein said four amino acid substitutions are a serine at position 1, a cysteine at position 149, an arginine at position 213 and a serine at position 238.  
   
   
       17 . The method of  claim 1  wherein said reporter group is a luminescent label.  
   
   
       18 . The method of  claim 17  wherein said luminescent label has an excitation wavelength of more than about 600 nanometers.  
   
   
       19 . The method of  claim 17  wherein said luminescent label has an emission wavelength of more than about 600 nanometers.  
   
   
       20 . The method of  claim 17  wherein said luminescent label is covalently coupled to said mutated glucose/galactose binding protein by reacting said mutated binding protein and at least one member selected from the group consisting of fluorescein, coumarins, rhodamines, 5TMRIA (tetramethylrhodamine-5-iodoacetamide), (9-(2(or 4)-(N-(2-maleimdylethyl)sulfonamidyl)-4(or 2)-sulfophenyl)-2,3,6,7,12,13,16,17-octahydro-(1H,5 11,11H,15Hxantheno(2,3,4-ij :5,6,7-i′j′)diquinolizin-18-ium salt) (Texas Red®), 2-(5-(1-(6-(N-(2maleimdylethyl)-amino)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1,3propyldienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium salt (Cy™3), N-((2-iodoacetoxy)ethyl)N-methyl)amino-7-nitrobenzoxadiazole (IANBD), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), pyrene, 6-amino-2,3-dihydro-2-(2-((iodoacetyl)amino)ethyl)-1,3-dioxo-1Hbenz(de)isoquinoline-5,8-disulfonic acid salt (lucifer yellow), 2-(5-(1-(6-(N-(2-maleimdylethyl)amino)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1,3-pentadienyl)-1ethyl-3,3-dimethyl-5-sulfo-3H-indolium salt (Cy™5), 4-(5-(4-dimethylaminophenyl)oxazol-2yl)phenyl-N-(2-bromoacetamidoethyl)sulfonamide (Dapoxyl®(2bromoacetamidoethyl)sulfonamide)), (N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diazas-indacene-2-yl)iodoacetamide (BODIPY® 507/545 IA), N-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N′-iodoacetylethylenediamine (BODIPY 530/550 IA), 5((((2-iodoacetyl)amino)ethyl) amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), and carboxyX-rhodamine, 5/6-iodoacetamide (XRIA 5,6).  
   
   
       21 . The method of  claim 1  additionally comprising the step of exposing said reporter group to an energy source capable of exciting said reporter group to emit said signal.