Methods and compositions for determination of glycated proteins

Abstract

This invention relates generally to the field of glycated protein detection. In particular, the invention provides chimeric proteins, nucleic acids encoding the chimeric proteins, methods and kits for assaying for a glycated protein in a sample, using inter alia, an amadoriase.

1 - 25 . (canceled) 
     
     
         26 : An isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein comprising, from N-terminus to C-terminus:
 a) a first peptidyl fragment comprising the amino acid sequence set forth in SEQ ID NO:1;   b) a second peptidyl fragment comprising an amadoriase; and   c) a third peptidyl fragment comprising the amino acid sequence set forth in SEQ ID NO:4.   
     
     
         27 . (canceled) 
     
     
         28 : The nucleic acid of  claim 26 , which comprises the nucleotide sequence set forth in SEQ ID NO:6 
       
         
           
                 
                 
               
                   (ATGGGAGGTTCGGGTGACGATGATGACCTGGCTCTCGCCGTCACTAAGT 
                     
                 
                     
                 
                   CATCATCTCTCCTGATCGTTGGTGCCGGGACTTGGGGCACCTCAACGGCT 
                 
                     
                 
                   CTGCACCTCGCGCGCCGCGGATATACCAACGTTACCGTGCTGGACCCCTA 
                 
                     
                 
                   TCCTGTCCCTAGCGCCATCTCCGCCGGAAACGACGTGAACAAAGTCATTA 
                 
                     
                 
                   GCAGTGGCCAATATTCGAATAACAAAGACGAAATCGAAGTGAATGAGATC 
                 
                     
                 
                   TTGGCGGAAGAGGCGTTTAACGGTTGGAAGAACGACCCGCTTTTCAAACC 
                 
                     
                 
                   GTATTATCATGATACGGGCCTGCTGATGTCTGCTTGCTCGCAGGAGGGCC 
                 
                     
                 
                   TGGATCGCCTGGGCGTCCGGGTACGTCCGGGCGAGGATCCTAATCTGGTG 
                 
                     
                 
                   GAACTTACCCGCCCGGAGCAATTTCGTAAACTGGCCCCGGAAGGCGTGTT 
                 
                     
                 
                   GCAAGGTGATTTTCCGGGTTGGAAAGGGTACTTTGCGCGTTCCGGCGCTG 
                 
                     
                 
                   GCTGGGCACATGCAAGGAATGCCTTAGTCGCAGCAGCACGCGAAGCACAG 
                 
                     
                 
                   CGCATGGGTGTAAAATTTGTTACTGGCACCCCGCAGGGTCGTGTAGTCAC 
                 
                     
                 
                   GTTAATCTTTGAAAATAACGATGTAAAAGGTGCCGTTACGGGCGATGGCA 
                 
                     
                 
                   AAATTTGGAGAGCGGAACGTACATTCCTGTGTGCTGGGGCTAGCGCGGGT 
                 
                     
                 
                   CAGTTCCTAGATTTCAAGAATCAACTTCGACCAACCGCTTGGACCCTGGT 
                 
                     
                 
                   ACACATTGCGTTAAAACCGGAAGAACGTGCGTTGTACAAAAATATACCGG 
                 
                     
                 
                   TTATCTTTAACATCGAACGGGGGTTTTTCTTTGAACCCGATGAGGAGCGC 
                 
                     
                 
                   GGTGAGATTAAAATATGCGATGAACACCCGGGCTACACAAATATGGTCCA 
                 
                     
                 
                   GAGTGCAGACGGCACGATGATGAGCATTCCGTTCGAAAAAACCCAGATTC 
                 
                     
                 
                   CAAAAGAAGCCGAAACGCGCGTTCGGGCCCTGCTGAAAGAGACAATGCCC 
                 
                     
                 
                   CAGCTGGCAGACCGTCCATTCAGCTTCGCACGCATTTGCTGGTGTGCCGA 
                 
                     
                 
                   TACCGCGAATCGCGAATTCCTGATACJATCGACATCCGCAGTACCACAGT 
                 
                     
                 
                   CTTGTGTTGGGCTGTGGTGCGAGCGGAAGAGGGTTTAAATATCTGCCTTC 
                 
                     
                 
                   TATTGGGAATCTCATTGTTGACGCGATGGAAGGTAAAGTGCCGCAAAAAA 
                 
                     
                 
                   TTCACGAATTAATCAAGTGGAACCCGGACATTGCGGCGAACCGTAACTGG 
                 
                     
                 
                   CGTGATACTCTGGGGCGTTTTGGCGGTCCAAATCGTGTGATGGATTTTCA 
                 
                     
                 
                   TGATGTGAAGGAATGGACCAATGTTCAGTATCGTGATATTTCCAAGCTGA 
                 
                     
                 
                   AAGGAGAGTTGGAAGGTaaGCCAATCCCTAACCCGTTACTGCGCACAGGC 
                 
                     
                 
                   CATCACCATCATCATCATTAA). 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         29 : An isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of  claim 26 . 
     
     
         30 : A recombinant cell containing the nucleic acid of  claim 26 . 
     
     
         31 : A method of producing a chimeric protein comprising growing a recombinant cell containing the nucleic acid of  claim 26  such that the encoded chimeric protein is expressed by the cell, and recovering the expressed chimeric protein. 
     
     
         32 - 86 . (canceled) 
     
     
         87 : The isolated nucleic acid of the  claim 26 , wherein the amadoriase is of  Aspergillus  sp. origin. 
     
     
         88 : The isolated nucleic acid of the  claim 26 , wherein the amadoriase uses FAD as a cofactor. 
     
     
         89 : The isolated nucleic acid of the  claim 26 , wherein the amadoriase has a FAD cofactor-binding consensus sequence Gly-X-Gly-X-X-Gly (SEQ ID NO:2), X being any amino acid residue. 
     
     
         90 : The isolated nucleic acid of the  claim 26 , wherein the amadoriase is selected from the group consisting of amadoriase Ia, amadoriase Ib, amadoriase Ic and amadoriase II. 
     
     
         91 : The isolated nucleic acid of the  claim 26 , wherein the amadoriase comprises the amino acid sequence set forth in SEQ ID NO:3 
       
         
           
                 
                 
               
                   (AVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDPYPVPSAISAGND 
                     
                 
                     
                 
                   VNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFKPYYHDTGLLMSA 
                 
                     
                 
                   CSQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGVLQGDFPGWKGYF 
                 
                     
                 
                   ARSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVVTLIFENNDVKGA 
                 
                     
                 
                   VTGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTLVHIALKPEERAL 
                 
                     
                 
                   YKNIPVIFNIERGFFFEPDEERGEIKICDEHPGYTNMVQSADGTMMSIPF 
                 
                     
                 
                   EKTQIPKEAETRVRALLKETMPQLADRPFSFARICWCADTANREFLIDRH 
                 
                     
                 
                   PQYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQKIHELIKWNPDIA 
                 
                     
                 
                   ANRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISKL). 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         92 : The isolated nucleic acid of the  claim 26 , wherein the chimeric protein further comprises, at its C-terminus, a fourth peptidyl fragment comprising a peptide tag. 
     
     
         93 : The isolated nucleic acid of the  claim 92 , wherein the peptide tag is selected from the group consisting of FLAG, HA, HA1, c-Myc, 6-His, AU1, EE, T7, 4A6, {acute over (ε)}, B, gE and Ty1 tag. 
     
     
         94 : The isolated nucleic acid of the  claim 26 , wherein the chimeric protein comprises the amino acid sequence set forth in SEQ ID NO:5 
       
         
           
                 
                 
               
                   (MGGSGDDDDLALAVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDP 
                     
                 
                     
                 
                   YPVPSAISAGNDVNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFK 
                 
                     
                 
                   PYYHDTGLLMSACSQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGV 
                 
                     
                 
                   LQGDFPGWKGYFARSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVV 
                 
                     
                 
                   TLIFENNDVKGAVTGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTL 
                 
                     
                 
                   VHIALKPEERALYKNIPVIFNIERGFFFEPDEERGEIKICDIEHPGYTNM 
                 
                     
                 
                   VQSADGTMMSIPFEKTQIPKEAETRVRALLKETMPQLADRPFSFARICWC 
                 
                     
                 
                   ADTANREFLIDRHPQYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQ 
                 
                     
                 
                   KIHELIKWNPDIAANRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISK 
                 
                     
                 
                   LKGELEGLPIPNPLLRTGHHHHHH). 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         95 : The isolated nucleic acid of the  claim 26 , wherein the first and the second peptidyl fragments are linked via a cleavable linkage.