Patent application title: Methods and compositions for determination of glycated proteins
Inventors:
Chong-Sheng Yuan (San Diego, CA, US)
Chong-Sheng Yuan (San Diego, CA, US)
Abhijit Datta (Carlsbad, CA, US)
Yuping Wang (San Diego, CA, US)
IPC8 Class: AC12P2104FI
USPC Class:
435 691
Class name: Chemistry: molecular biology and microbiology micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition recombinant dna technique included in method of making a protein or polypeptide
Publication date: 2008-10-02
Patent application number: 20080241880
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Patent application title: Methods and compositions for determination of glycated proteins
Inventors:
Chong-Sheng Yuan
Abhijit Datta
Yuping Wang
Agents:
MORRISON & FOERSTER LLP
Assignees:
Origin: PALO ALTO, CA US
IPC8 Class: AC12P2104FI
USPC Class:
435 691
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.Claims:
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; andc) 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 TABLE-US-00009 (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 TABLE-US-00010 (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 TABLE-US-00011 (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.
Description:
BACKGROUND OF THE INVENTION
[0001]A glycated protein is a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. See e.g., U.S. Pat. No. 6,127,138. Such a non-enzymatic and irreversible binding reaction is also called "Amadori rearrangement," and therefore the above-mentioned glycated protein may also be called "Amadori compound" in some cases.
[0002]Nonenzymatic glycation of proteins has been implicated in the development of certain diseases, e.g., diabetic complications and the aging process (Takahashi et al., J. Biol. Chem., 272(19):12505-7 (1997); and Baynes and Monnier, Prog. Clin. Biol. Res., 304:1-410 (1989)). This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links. Considerable interest has focused on the Amadori product that is the most important "early" modification during nonenzymatic glycation in vitro and in vivo.
[0003]Various assays for glycated proteins are known. For example, U.S. Pat. No. 6,127,138 discloses that a sample containing a glycated protein is treated with Protease XIV or a protease from Aspergillus genus, thereafter (or while treating the sample with the above protease) FAOD (fructosyl amino acid oxidase) is caused to react with the sample so as to measure the amount of oxygen consumed by the FAOD reaction or the amount of the resultant reaction product, thereby to measure the glycated protein.
[0004]In another example, U.S. Pat. No. 6,008,006 discloses that the amount of glycated proteins in a sample can be quantified by reacting the sample with first a reagent which is a combination of a protease and a peroxidase and second with a ketoamine oxidase. U.S. Pat. No. 6,008,006 also discloses a kit which contains the combined peroxidase/protease enzyme reagent and also the ketoamine oxidase.
BRIEF SUMMARY OF THE INVENTION
[0005]In one aspect, the present invention is directed to an isolated chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase.
[0006]In another aspect, the present invention is directed to an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase. Recombinant cells comprising the nucleic acid and methods for producing the chimeric protein using the nucleic acid are also provided.
[0007]In still another aspect, the present invention is directed to a method for assaying for a glycated protein in a sample, which method comprises: a) contacting a sample to be assayed with a protease to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) contacting said generated glycated peptide or glycated amino acid with a chimeric protein comprising, from N-terminus to C-terminus: i) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and ii) a second peptidyl fragment comprising an amadoriase, to oxidize said glycated peptide or glycated amino acid; and c) assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.
[0008]In yet another aspect, the present invention is directed to a kit for assaying for a glycated protein in a sample, which kit comprises: a) a protease to generate glycated peptide or glycated amino acid from a glycated protein, if contained in a sample; b) the above-described chimeric protein to oxidize said glycated peptide or glycated amino acid; and c) means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.
[0009]In yet another aspect, the present invention is directed to a method for assaying for a glycated protein in a sample, which method comprises: a) contacting a sample to be assayed with a proteinase K to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) contacting said generated glycated peptide or glycated amino acid with an amadoriase to oxidize said glycated peptide or glycated amino acid; and c) assessing oxidation of said glycated peptide or glycated amino acid by said amadoriase to determine the presence and/or amount of said glycated protein in said sample.
[0010]In yet another aspect, the present invention is directed to a kit for assaying for a glycated protein in a sample, which kit comprises: a) a proteinase K to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) an amadoriase to oxidize said glycated peptide or glycated amino acid; and c) means for assessing oxidation of said glycated peptide or glycated amino acid by said amadoriase to determine the presence and/or amount of said glycated protein in said sample.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0011]FIG. 1 illustrates a comparison between an exemplary GSP kit and a Randox Fructosamine kit.
[0012]FIG. 2 illustrates assay linearity of an exemplary method for assaying for a glycated protein in a sample.
[0013]FIGS. 3 and 4 show the dose-dependent reaction with fructosyl-valine.
[0014]FIG. 5 shows the dose-dependent signal with patient hemoglobin digested with a proteinase (5 min. digestion).
DETAILED DESCRIPTION OF THE INVENTION
[0015]For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.
A. DEFINITIONS
[0016]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
[0017]As used herein, "a" or "an" means "at least one" or "one or more."
[0018]As used herein, a "leader sequence" refers to a peptide sequence, when fused to a target peptide or protein, increases stability and/or expression level of the target peptide or protein. Normally, a leader sequence increases stability and/or expression level of the target peptide or protein for at least 50%. Preferably, a leader sequence increases stability and/or expression level of the target peptide or protein for at least 1 fold, 2 folds, 5 folds, 10 folds or more than 10 folds. In the regulation of gene expression for enzymes concerned with amino acid synthesis in prokaryotes, the leader sequence codes for the leader peptide that contains several residues of the amino acid being regulated. Transcription is closely linked to translation, and if translation is retarded by limited supply of aminoacyl tRNA for the specific amino acid, the mode of transcription of the leader sequence permits full transcription of the operon genes; otherwise complete transcription of the leader sequence prematurely terminates transcription of the regulated gene.
[0019]As used herein, a "glycated protein" refers to a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. See e.g., U.S. Pat. No. 6,127,138. Such a non-enzymatic and irreversible binding reaction is also called "Amadori rearrangement," and therefore the above-mentioned glycated protein may also be called "Amadori compound" in some cases.
[0020]As used herein, an "amadoriase" refers to an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2, as shown in the following reaction:
R1--CO--CH2--NH--R2+O2+H2O→R1--CO--CH- O+R2--NH2+H2O2
[0021]wherein, R1 represents the aldose residue of a reducing sugar and R2 represents a residue of an amino acid, protein or peptide. Other synonyms of amadoriase include fructosyl amino acid oxidase (FAOD) and fructosyl amine:oxygen oxidoreductase (FAOO). For purposes herein, the name "amadoriase" is used herein, although all such chemical synonyms are contemplated. "Amadoriase" also encompasses a functional fragment or a derivative that still substantially retain its enzymatic activity catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2. Typically, a functional fragment or derivative retains at least 50% of its amadoriase activity. Preferably, a functional fragment or derivative retains at least 60%, 70%, 80%, 90%, 95%, 99% or 100% of its amadoriase activity. It is also intended that an amadoriase can include conservative amino acid substitutions that do not substantially alter its activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson, et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. Co., p. 224). Such exemplary substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:
TABLE-US-00001 TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu
Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.
[0022]As used herein, "glycohemoglobin" refers to a fructosylamine derivative produced by the glycation of hemoglobin in blood.
[0023]As used herein, "glycoalbumin" refers to a fructosylamine derivative produced by the glycation of albumin in blood.
[0024]As used herein, "fructosamine" refers to a derivative (having a reducing ability) produced by the glycation of a protein in blood.
[0025]As used herein, a "composition" refers to any mixture of two or more products or compounds. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous, or any combination thereof.
[0026]As used herein, a "combination" refers to any association between two or among more items.
[0027]As used herein, "plasma" refers to the fluid, noncellular portion of the blood, distinguished from the serum obtained after coagulation.
[0028]As used herein, "serum" refers to the fluid portion of the blood obtained after removal of the fibrin clot and blood cells, distinguished from the plasma in circulating blood.
[0029]As used herein, "fluid" refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.
[0030]As used herein, "peroxidase" refers to an enzyme that catalyses a host of reactions in which hydrogen peroxide is a specific oxidizing agent and a wide range of substrates act as electron donors. It is intended to encompass a peroxidase with conservative amino acid substitutions that do not substantially alter its activity. The chief commercially available peroxidase is horseradish peroxidase.
[0031]As used herein, "glucose oxidase" refers to an enzyme that catalyzes the formation of gluconic acid and H2O2 from glucose, H2O and O2. It is intended to encompass glucose oxidase with conservative amino acid substitutions that do not substantially alter its activity.
[0032]As used herein, "hexokinase" refers to an enzyme that catalyses the transfer of phosphate from ATP to glucose to form glucose-6-phosphate, the first reaction in the metabolism of glucose via the glycolytic pathway. It is intended to encompass hexokinase with conservative amino acid substitutions that do not substantially alter its activity.
[0033]As used herein, the abbreviations for any protective groups, amino acids and other compounds, are in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature, unless otherwise indicated (see Biochemistry 11: 1726 (1972)).
B. CHIMERIC PROTEINS COMPRISING AN AMADORIASE AND NUCLEIC ACIDS ENCODING THE SAME
[0034]In one aspect, the present invention is directed to an isolated chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase.
[0035]Any suitable bacterial leader sequences can be used. As disclosed in U.S. Pat. No. 6,194,200, expression of the polypeptide of interest as a fused protein with a leader sequence from another gene has several advantages in addition to providing for stability. For example, the presence of the N-terminal amino acids provides a means for using general purification techniques for purification of any of a variety of polypeptides. For example, the N-terminal amino acids of the N-protein are predictably antigenic, and thus specific antibodies raised against the N-terminal amino acids of the N-protein may be used for the amino purification of the fusion proteins containing the N-terminus of the N-protein. Furthermore, the N-terminus of the N-protein has a high positive charge, which facilitates purification of the desired protein by ion-exchange chromatography, and the like.
[0036]The leader sequence can also be a hydrophobic amino acid sequence, which may additionally function as a signal sequence for secretion. See U.S. Pat. No. 6,194,200. A DNA sequence encoding the signal sequence is joined upstream from and in reading frame with the gene of interest. Typically, the signal sequence includes a cleavage site which is recognized by a signal sequence peptidase. Thus, positioning the polypeptide of interest directly after the signal sequence cleavage site will allow it to be specifically cleaved from the signal sequence and secreted as a mature polypeptide. Examples of hydrophobic amino acid sequences include the bacterial alkaline phosphatase signal sequence; the OMP-A, B, C, D, E or F signal sequences; the LPP signal sequence, β-lactamase signal sequence; and toxin signal sequences.
[0037]Other leader sequences which can be used include hydrophilic sequences, for example the N-terminal 41 amino acid residues from amphiregulin which may provide for modification of the function of the polypeptide of interest. See U.S. Pat. No. 6,194,200. In addition, a cytotoxic agent such as a toxin A-chain fragment, ricin A-chain, snake venom growth arresting peptide, or a targeting molecule such as a hormone or antibody can be coupled covalently with the leader sequence with in most cases minimal effect on the biological activity of the gene product of interest. As with the other leader sequences, a DNA sequence encoding the leader sequence is joined upstream from and in reading frame with the gene of interest.
[0038]Where the leader sequence is not a signal sequence or does not contain a convenient natural cleavage site, additional amino acids may be inserted between the gene of interest and the leader sequence to provide an enzymatic or chemical cleavage site for cleavage of the leader peptide, following purification of the fusion protein, to allow for subsequent purification of the mature polypeptide. See U.S. Pat. No. 6,194,200. For example, introduction of acid-labile aspartyl-proline linkages between the two segments of the fusion protein facilitates their separation at low pH. This method is not suitable if the desired polypeptide is acid-labile. The fusion protein may be cleaved with, for example, cyanogen bromide, which is specific for the carboxy side of methionine residues. Positioning a methionine between the leader sequence and the desired polypeptide would allow for release of the desired polypeptide. This method is not suitable when the desired polypeptide contains methionine residues.
[0039]Other bacterial leader sequences disclosed in the following patents, patent application and references can also be used: WO 00/28041 and WO 89/03886; U.S. Pat. Nos. 5,914,250, 5,885,811, 5,171,670, 5,030,563, 4,948,729 and 4,588,684; EP Patent Nos. EP 0,196,864, EP 0,186,643 and EP 0,121,352; Michiels et al., Trends Microbiol., 9(4):164-8 (2001); Hobom et al., Dev. Biol. Stand, 84:255-62 (1995); Hardy and Randall, J. Cell. Sci. Suppl., 11:29-43 (1989); Saier et al., FASEB J, 2(3):199-208 (1988); and Peakman et al., Nucleic Acids Res., 20(22):6111-2 (1992). Preferably, the bacterial leader sequence is a leader sequence of an E. coli. protein, e.g., the E. coli. leader sequences disclosed in Roesser and Yanofsky, Nucleic Acids Res., 19(4):795-800 (1991); and Kuhn et al., Mol. Gen. Genet., 167(3):235-41 (1979).
[0040]In one example, the leader sequence has at least 40% identity to the amino acid sequence set forth in SEQ ID NO:1 (MGGSGDDDDLAL), in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:1. Preferably, the leader sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:1, in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:1. Also preferably, the leader sequence binds to an antibody that specifically binds to an amino acid sequence set forth in SEQ ID NO:1. Still preferably, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO:1.
[0041]The first peptidyl fragment can have any suitable length. For example, the first peptidyl fragment comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. Preferably, the first peptidyl fragment comprises about 20 amino acid residues.
[0042]Any suitable amadoriase can be used. In one example, the amadoriase is of Aspergillus sp. origin (See e.g., Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997)). In another example, the amadoriase uses FAD as a cofactor. Preferably, 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. In still another example, the amadoriase is amadoriase Ia, amadoriase Ib, amadoriase Ic or amadoriase II (See e.g., Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997)). Amino acid sequence homology between the N-terminal sequence of amadoriases Ia, amadoriase Ib, amadoriase Ic and amadoriase II is shown in the following Table 2. These data were obtained by a computerized search using the combined GenBank® CDS translations/PDB/SwissProt/SPupdate/PIR data base (See Table IV of Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997)). Numbers indicate the amino acid positions within each sequence. Conservative substitutions are indicated by (+). The data of amadoriases correspond to the N-terminal sequences obtained in Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997).
TABLE-US-00002 TABLE 2 Amino acid sequence homology between the N- terminal sequence of amadoriase Ia, amadoriase Ib, amadoriase Ic and amadoriase II Sequences with Protein residue nos. SEQ ID NO Amadoriases Ia 1A P S I L S T E S S I SEQ ID NO:7 (C/T) V I G A G T W G20 Amadoriase Ib 1A P S I L S T E S S I SEQ ID NO:8 I V I G A G T W G20 Amadoriase Ic 1S T E S S I I V I G SEQ ID NO:9 A G T W G (C) (S) T A L20 Amadoriase II 1A V T K S S S L L I SEQ ID NO:10 V G A G T W G T S T20
[0043]Other amadoriases, e.g., amadoriases disclosed in GenBank Accession No. U82830 (Takahashi et al., J. Biol. Chem., 272(19):12505-12507 (1997) and amadoriases disclosed U.S. Pat. No. 6,127,138 can also be used. A functional fragment or a derivative of an amadoriase that still substantially retain its enzymatic activity catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2 can also be used.
[0044]Normally, a functional fragment or a derivative of an amadoriase retain at least 50% of its enzymatic activity. Preferably, a functional fragment or a derivative of an amadoriase retain at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of its enzymatic activity.
[0045]Assays for enzymatic activities of amadoriases are known in the art (See e.g., Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997) and U.S. Pat. No. 6,127,138). Four exemplary assays for enzymatic activities of amadoriases are disclosed in Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997).
[0046]Glucosone Formation
[0047]In this assay, the enzyme activity is monitored by the release of glucosone measured by a colorimetric reaction with OPD using fructosyl propylamine as a substrate. This assay is based on the end point measurement of glucosone formed after 120 min of reaction time. The reaction mixture contains 20 mM sodium phosphate, pH 7.4, 10 mM OPD, 10 mM fructosyl propylamine, and enzyme protein in a final volume of 1 ml. After incubation at 37° C. for 2 h, the absorbance at 320 nm is measured. The reaction is linear to 240 min in a dose-dependent manner under these conditions. One unit of enzyme activity is defined as the amount of the enzyme that produces 1 μmol of glucosone/min. Synthesized glucosone is used as a standard.
[0048]Free Amine Assay
[0049]To assay the release of free amine, fluorescence is measured after reaction with fluorescamine. Twenty-five (25) μl of a solution of pure enzyme or enzyme-rich fraction, 15 μl of 20% fructosyl propylamine in water, and 250 μl of PBS are incubated at 37° C. for different times as indicated. The reaction is stopped by filtration through a Microcon-10 (Amicon, Beverly, Mass.) at 4 C. One (1) μl of the pure or 1:10 diluted filtrate is added to 1.5 ml of 50 mM phosphate buffer pH 8.0. Under vigorous vortexing 0.5 ml of 0.03% fluorescamine in dioxane is rapidly added. After 5 min fluorescence is measured (λex=390 nm, λem=475 nm). A standard plot is made with 6-150 ng of propylamine.
[0050]H2O2 Assay
[0051]Hydrogen peroxide is quantitated by the quinone dye assay according to Sakai et al., Biosci. Biotech. Biochem., 59:487-491 (1995). The reaction mixture contains 20 mM Tris-HCl, pH 8.0, 1.5 mM 4-aminoantipyrine, 2.0 mM phenol, 2.0 units of peroxidase, 10 mM fructosyl propylamine, and enzyme protein in a total volume of 1 ml. Production of the H2O2 is monitored by the formation of a quinone dye following the absorbance at 505 nm (ε=5.13×103). The production of 0.5 μmol of quinone dye corresponds to the formation of 1.0 μmol of H2O2.
[0052]Oxygen Consumption
[0053]Oxygen consumption is determined with a YSI-Beckman glucometer II equipped with a Clarke type oxygen electrode as described in Gerhardinger, et al., J. Biol. Chem., 270:218-224 (1995). Briefly, enzyme (50 μl) is added to the chamber containing 750 μl of PBS and 650 μl of water. The reaction is started by addition of 50 μl of 300 mM fructosyl propylamine (final concentration 10 mM).
[0054]In another example, the amadoriase has at least 40% identity to the amino acid sequence set forth in SEQ ID NO:3
TABLE-US-00003 (AVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDPYPVPSAISAGND VNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFKPYYHDTGLLMSA SQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGVLQGDFPGWKGYFA RSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVVTLIFENNDVKGAV TGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTLVHIALKPEERALY KNIPVIFNIERGFFFEPDEERGEIKICDEHPGYTNMVQSADGTMMSIPFE KTQIPKEAETRVRALLKETMPQLADRPFSFARICWCADTANREFLIDRHP QYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQKIHELIKWNPDIAA NRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISKL),
in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:3. Preferably, the amadoriase has at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:3, in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:3. Also preferably, the amadoriase binds to an antibody that specifically binds to an amino acid sequence set forth in SEQ ID NO:3. Also preferably, the amadoriase comprises the amino acid sequence set forth in SEQ ID NO:3.
[0055]The first and second peptidyl fragments can be linked via any suitable linkage. For example, the first and second peptidyl fragments can be linked via a cleavable linkage.
[0056]The isolated chimeric protein can further comprise, at its C-terminus, a third peptidyl fragment comprising a second bacterial leader sequence from about 5 to about 30 amino acid residues. Any suitable bacterial leader sequences, including the ones described above, can be used.
[0057]In one example, the second bacterial leader sequence is a leader sequence of an E. coli. protein. in another example, the second bacterial leader sequence has at least 40% identity to the amino acid sequence set forth in SEQ ID NO:4 (KGELEGLPIPNPLLRTG), in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:4. Preferably, the second bacterial leader sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:4, in which the percentage identity is determined over an amino acid sequence of identical size to the amino acid sequence set forth in SEQ ID NO:4. Also preferably, the second bacterial leader sequence binds to an antibody that specifically binds to an amino acid sequence set forth in SEQ ID NO:4. Also preferably, the second bacterial leader sequence comprises the amino acid sequence set forth in SEQ ID NO:4.
[0058]The third peptidyl fragment can have an suitable length. For example, the third peptidyl fragment comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. Preferably, the third peptidyl fragment comprises about 20 amino acid residues.
[0059]The isolated chimeric protein of can further comprise, at its C-terminus, a third peptidyl fragment comprising a peptide tag. Any suitable tag can be used. For example, the tag can be FLAG, HA, HA1, c-Myc, 6-His, AU1, EE, T7, 4A6, ε, B, gE and Ty1 tag (See Table 3).
TABLE-US-00004 TABLE 3 Exemplary epitope tag systems SEQ Epitope Peptide ID Antibody Reference FLAG AspTyrLysAspAspAspLys 11 4E11 Prickett1 HA TyrProTyrAspValPRoAspTyrAla 12 12Ca5 Xie2 HA1 CysGlnAspLeuProGlyAsnAspAsnSerThr 13 mouse MAb Nagelkerken3 c-Myc GluGlnLysLeuIleSerGluGluAspLeu 14 9E10 Xie2 6-His HisHisHisHisHisHis 15 BAbCO* AU1 AspThrTyrArgTyrIle 16 BAbCO EE GluTyrMetProMetGlu 17 anti-EE Tolbert4 T7 AlaSerMetThrGlyGlyGlnGlnMetGlyArg 18 Invitrogen Chen5 Tseng6 4A6 SerPheProGlnPheLysProGlnGluIle 19 4A6 Rudiger7 ε LysGlyPheSerTyrPheGlyGluAspLeuMetPro 20 anti-PKCε Olah8 B GlnTyrProAlaLeuThr 21 D11, F10 Wang9 gE GlnArgGlnTyrGlyAspValPheLysGlyAsp 22 3B3 Grose10 Ty1 GluValHisThrAsnGlnAspProLeuAsp 23 BB2, TYG5 Bastin11 1Prickett, et al., BioTechniques, 7(6): 580-584 (1989) 2Xie, et al., Endocrinology, 139(11): 4563-4567 (1998) 3Nagelkerke, et al., Electrophoresis, 18: 2694-2698 (1997) 4Tolbert and Lameh, J. Neurochem., 70: 113-119 (1998) 5Chen and Katz, BioTechniques, 25(1): 22-24 (1998) 6Tseng and Verma, Gene, 169: 287-288 (1996) 7Rudiger, et al., BioTechniques, 23(1): 96-97 (1997) 8Olah, et al., Biochem., 221: 94-102 (1994) 9Wang, et al., Gene, 169(1): 53-58 (1996) 10Grose, U.S. Pat. No. 5,710,248 11Bastin, et al., Mol. Biochem. Parasitology, 77: 235-239 (1996) Invitrogen, Sigma, Santa Cruz Biotech
[0060]In an example, the isolated chimeric protein comprises the amino acid sequence set fourth in SEQ ID NO:5
TABLE-US-00005 (MGGSGDDDDLALAVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDP YPVPSAISAGNDVNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFK PYYHDTGLLMSACSQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGV LQGDFPGWKGYFARSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVV TLIFENNDVKGAVTGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTL VHIALKPEERALYKNIPVIFNIERGFFFEPDEERGEIKICDEHPGYTNMV QSADGTMMSIPFEKTQIPKEAETRVRALLKETMPQLADRPFSFARICWCA DTANREFLIDRHPQYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQK IHELIKWNPDIAANRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISKL KGELEGLPIPNPLLRTGHHHHHH).
[0061]In another aspect, the present invention is directed to an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase.
[0062]In one example, the isolated nucleic acid comprises a nucleotide sequence encoding the chimeric protein comprising the amino acid sequence set forth in SEQ ID NO:5. In another example, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO:6
TABLE-US-00006 (ATGGGAGGTTCGGGTGACGATGATGACCTGGCTCTCGCCGTCACTAAGT CATCATCTCTCCTGATCGTTGGTGCCGGGACTTGGGGCACCTCAACGGCT CTGCACCTCGCGCGCCGCGGATATACCAACGTTACCGTGCTGGACCCCTA TCCTGTCCCTAGCGCCATCTCCGCCGGAAACGACGTGAACAAAGTCATTA GCAGTGGCCAATATTCGAATAACAAAGACGAAATCGAAGTGAATGAGATC TTGGCGGAAGAGGCGTTTAACGGTTGGAAGAACGACCCGCTTTTCAAACC GTATTATCATGATACGGGCCTGCTGATGTCTGCTTGCTCGCAGGAGGGCC TGGATCGCCTGGGCGTCCGGGTACGTCCGGGCGAGGATCCTAATCTGGTG GAACTTACCCGCCCGGAGCAATTTCGTAAACTGGCCCCGGAAGGCGTGTT GCAAGGTGATTTTCCGGGTTGGAAAGGGTACTTTGCGCGTTCCGGCGCTG GCTGGGCACATGCAAGGAATGCCTTAGTGGCAGCAGCACGCGAAGCACAG CGCATGGGTGTAAAATTTGTTACTGGCACCCCGCAGGGTCGTGTAGTCAC GTTAATCTTTGAAAATAACGATGTAAAAGGTGCCGTTACGGGCGATGGCA AAATTTGGAGAGCGGAACGTACATTCCTGTGTGCTGGGGCTAGCGCGGGT CAGTTCCTAGATTTCAAGAATCAACTTCGACCAACCGCTTGGACCCTGGT ACACATTGCGTTAAAACCGGAAGAACGTGCGTTGTACAAAAATATACCGG TTATCTTTAACATCGAACGGGGGTTTTTCTTTGAACCCGATGAGGAGCGC GGTGAGATTAAAATATGCGATGAACACCCGGGCTACACAAATATGGTCCA GAGTGCAGACGGCACGATGATGAGCATTCCGTTCGAAAAAACCCAGATTC CAAAAGAAGCCGAAACGCGCGTTCGGGCCCTGCTGAAAGAGACAATGCCC CAGCTGGCAGACCGTCCATTCAGCTTCGCACGCATTTGCTGGTGTGCCGA TACCGCGAATCGCGAATTCCTGATAGATCGACATCCGCAGTACCACAGTC TTGTGTTGGGCTGTGGTGCGAGCGGAAGAGGGTTTAAATATCTGCCTTCT ATTGGGAATCTCATTGTTGACGCGATGGAAGGTAAAGTGCCGCAAAAAAT TCACGAATTAATCAAGTGGAACCCGGACATTGCGGCGAACCGTAACTGGC GTGATACTCTGGGGCGTTTTGGCGGTCCAAATCGTGTGATGGATTTTCAT GATGTGAAGGAATGGACCAATGTTCAGTATCGTGATATTTCCAAGCTGAA AGGAGAGTTGGAAGGTaaGCCAATCCCTAACCCGTTACTGCGCACAGGCC ATCACCATCATCATCATTAA).
[0063]In still another example, the isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence encoding a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase.
[0064]A recombinant cell containing the nucleic acid, or a complementary strand thereof, encoding a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase, is contemplated.
[0065]A method of producing a chimeric protein is also contemplated, which method comprising growing a recombinant cell containing the nucleic acid encoding a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase, such that the encoded chimeric protein is expressed by the cell, and recovering the expressed chimeric protein. The product of the method is further contemplated.
[0066]The chimeric proteins and the nucleic acids encoding the chimeric proteins can be prepared by any suitable methods, e.g., chemical synthesis, recombinant production or a combination thereof (See e.g., Current Protocols in Molecular Biology, Ausubel, et al. eds., John Wiley & Sons, Inc. (2000) and Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989)).
C. METHODS AND KITS FOR ASSAYING FOR A GLYCATED PROTEIN USING A CHIMERIC PROTEIN
[0067]In still another aspect, the present invention is directed to a method for assaying for a glycated protein in a sample, which method comprises: a) contacting a sample to be assayed with a protease to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) contacting said generated glycated peptide or glycated amino acid with a chimeric protein comprising, from N-terminus to C-terminus: i) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and ii) a second peptidyl fragment comprising an amadoriase, to oxidize said glycated peptide or glycated amino acid; and c) assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.
[0068]The present methods can be used to assay any suitable sample. Preferably, the sample is a blood sample, e.g., a plasma, serum, red blood cell or whole blood sample.
[0069]The present methods can be used to assay any suitable glycated proteins. Preferably, the glycated protein to be assayed is glycoalbumin or glycohemoglobin.
[0070]Any suitable protease can be used in the present methods. Either an endo-type protease or an exo-type protease can be used. Exemplary endo-type proteases include trypsin, α-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XVII, protease XXI, lysyl-endopeptidase, prolether and bromelain F. Exemplary exo-type proteases include an aminopeptidase or a carboxypeptidase. In one example, the protease is proteinase K, pronase E, ananine, thermolysin, subtilisin or cow pancreas proteases.
[0071]The protease can be used to generates a glycated peptide of any suitable size. For example, the protease can be used to generates a glycated peptide from about 2 to about 30 amino acid residues. In another example, the protease is used to generate glycated glycine, glycated valine or glycated lysine residue or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue.
[0072]Any suitable chimeric proteins, including the ones described in the above Section B, can be used in the present methods. In one example, the chimeric protein comprises the amino acid sequence set forth in SEQ ID NO:5. In another example, the chimeric protein is encoded by the nucleotide sequence set forth in SEQ ID NO:6.
[0073]The oxidation of the glycated peptide or glycated amino acid can be assessed by any suitable methods. For example, the oxidation of the glycated peptide or glycated amino acid can be assessed by assessing consumption of the glycated peptide or glycated amino acid, H2O or O2 in the oxidation reaction or the formation of the oxidized glucose (glucosone), H2O2 or the amino acid in the oxidation reaction.
[0074]The O2 consumption by can be assessed by any suitable methods. For example, the O2 consumption can be assessed by an oxygen electrode.
[0075]The H2O2 formation can be assessed by any suitable methods. For example, the H2O2 formation can be assessed by a peroxidase. Any peroxidase can be used in the present methods. More preferably, a horseradish peroxidase is used. For example, the horseradish peroxidases with the following GenBank accession Nos. can be used: E01651; D90116 (prxC3 gene); D90115 (prxC2 gene); J05552 (Synthetic isoenzyme C(HRP-C)); S14268 (neutral); OPRHC (C1 precursor); S00627 (C1C precursor); JH0150 (C3 precursor); S00626 (C1B precursor); JH0149 (C2 precursor); CAA00083 (Armoracia rusticana); and AAA72223 (synthetic horseradish peroxidase isoenzyme C(HRP-C)). In another example, the H2O2 formation can be assessed by a peroxidase and Trinder reaction. The glycated peptide or glycated amino acid can be contacted with the chimeric protein and the peroxidase sequentially or simultaneously.
[0076]The glucosone formation can be assessed by any suitable methods. For example, the glucosone formation can be assessed by a glucose oxidase. Any suitable glucose oxidase can be used. For example, glucose oxidases encoded by the nucleotide sequences with the following GenBank accession Nos. can be used: AF012277 (Penicillium amagasakiense); U56240 (Talaromyces flavus); X16061 (Aspergillus niger gox gene); X56443 (A. niger god gene); J05242 (A. niger); AF012277 (Penicillium amagasakiense); U56240 (Talaromyces flavus); X16061 (Aspergillus niger gox gene); X56443 (A. niger god gene); J05242 (A. niger glucose). Preferably, the nucleotide sequences with the GenBank accession No. J05242 (See also Frederick, et al., J. Biol. Chem., 265(7):3793-802 (1990)) and the nucleotide sequences described in U.S. Pat. No. 5,879,921 can be used in obtaining nucleic acid encoding glucose oxidase.
[0077]In another example, the glucosone formation can be assessed by a combination of glucose 6-phosphate dehydrogenase and hexokinase. Any suitable glucose 6-phosphate dehydrogenase can be used. For example, glucose 6-phosphate dehydrogenase disclosed in the following patents and patent applications can be used: WO 03/042389, WO 01/98472, WO 93/06125, and U.S. Pat. Nos. 6,127,345, 6,069,297, 5,856,104, 5,308,770, 5,244,796, 5,229,286, 5,137,821 and 4,847,195. Any suitable hexokinase can be used. For example, hexokinase disclosed in the following patents and patent applications can be used: WO 02/20795, US2002/009779, WO 01/90378, WO 01/90325, WO 01/68694, WO 01/47968 and U.S. Pat. No. 5,948,665.
[0078]If desirable, the protease can be inactivated before or current with the contact between the glycated peptide or glycated amino acid and the chimeric protein. The protease can be inactivated by any suitable methods. For example, the protease can be inactivated by a heat treatment or an inhibitor of the protease.
[0079]If desirable, interference of the assay can be countered. For example, ascorbate interference can be countered using a copper (II) compound, a cholic acid or a bathophenanthroline disulphonic acid or a mixture thereof. Bilirubin interference can be countered using a ferrocyanide salt.
[0080]The present methods can be used for any suitable purpose. Preferably, the method used in the prognosis or diagnosis of a disease or disorder, e.g., diabetes.
[0081]In yet another aspect, the present invention is directed to a kit for assaying for a glycated protein in a sample, which kit comprises: a) a protease to generate glycated peptide or glycated amino acid from a glycated protein, if contained in a sample; b) a chimeric protein comprising, from N-terminus to C-terminus: i) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and ii) a second peptidyl fragment comprising an amadoriase, to oxidize said glycated peptide or glycated amino acid; and c) means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.
[0082]Any suitable means can be included in the present kits. For example, the means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein can comprise a peroxidase. Preferably, the chimeric protein and the peroxidase are formulated in a single composition.
D. METHODS AND KITS FOR ASSAYING FOR A GLYCATED PROTEIN USING PROTEINASE K AND AN AMADORIASE
[0083]In yet another aspect, the present invention is directed to a method for assaying for a glycated protein in a sample, which method comprises: a) contacting a sample to be assayed with a proteinase K to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) contacting said generated glycated peptide or glycated amino acid with an amadoriase to oxidize said glycated peptide or glycated amino acid; and c) assessing oxidation of said glycated peptide or glycated amino acid by said amadoriase to determine the presence and/or amount of said glycated protein in said sample.
[0084]The present methods can be used to assay any suitable sample. Preferably, the sample is a blood sample, e.g., a plasma, serum, red blood cell or whole blood sample.
[0085]The present methods can be used to assay any suitable glycated proteins. Preferably, the glycated protein to be assayed is glycoalbumin or glycohemoglobin.
[0086]Any suitable amadoriase, including the ones described in the above Sections B and C, can be used in the present methods. For example, the amadoriase can comprise a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase. Preferably, the chimeric protein comprises the amino acid sequence set forth in SEQ ID NO:5. Also preferably, the chimeric protein is encoded by the nucleotide sequence set forth in SEQ ID NO:6.
[0087]The oxidation of the glycated peptide or glycated amino acid can be assessed by any suitable methods. For example, the oxidation of the glycated peptide or glycated amino acid can be assessed by assessing consumption of the glycated peptide or glycated amino acid, H2O or O2 in the oxidation reaction or the formation of the oxidized glucose (glucosone), H2O2 or the amino acid in the oxidation reaction.
[0088]The O2 consumption can be assessed by any suitable methods. For example, the O2 consumption can be assessed by an oxygen electrode.
[0089]The H2O2 formation can be assessed by any suitable methods. For example, the H2O2 formation can be assessed by a peroxidase. Any peroxidase, including the ones described in the above Sections C, can be used in the present methods. More preferably, a horseradish peroxidase is used. In another example, the H2O2 formation can be assessed by a peroxidase and Trinder reaction. The glycated peptide or glycated amino acid can be contacted with the chimeric protein and the peroxidase sequentially or simultaneously.
[0090]The glucosone formation can be assessed by any suitable methods. For example, the glucosone formation can be assessed by a glucose oxidase. Any suitable glucose oxidase, including the ones described in the above Sections C, can be used.
[0091]In another example, the glucosone formation can be assessed by a combination of glucose 6-phosphate dehydrogenase and hexokinase. Any suitable glucose 6-phosphate dehydrogenase, including the ones described in the above Sections C, can be used. Any suitable hexokinase, including the ones described in the above Sections C, can be used.
[0092]Any suitable proteinase K can be used in the present methods. For example, proteinase K disclosed in the following patents and patent applications can be used: WO 02/072634, WO 02/064760, WO 96/28556, and U.S. Pat. Nos. 6,451,574 and 5,344,770. Preferably, proteinase K from Tritirachium album is used (See e.g., Sigma-Aldrich Catalog No. 82452).
[0093]If desirable, the proteinase K can be inactivated before or concurrent with the contact between the glycated peptide or glycated amino acid and the amadoriase. For example, the proteinase K can be inactivated by a heat treatment or an inhibitor of the proteinase K.
[0094]If desirable, interference of the assay can be countered. For example, ascorbate interference can be countered using a copper (II) compound, a cholic acid or a bathophenanthroline disulphonic acid or a mixture thereof. Bilirubin interference can be countered using a ferrocyanide salt.
[0095]The present methods can be used for any suitable purpose. Preferably, the method used in the prognosis or diagnosis of a disease or disorder, e.g., diabetes.
[0096]In yet another aspect, the present invention is directed to a kit for assaying for a glycated protein in a sample, which kit comprises: a) a proteinase K to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample; b) an amadoriase to oxidize said glycated peptide or glycated amino acid; and c) means for assessing oxidation of said glycated peptide or glycated amino acid by said amadoriase to determine the presence and/or amount of said glycated protein in said sample.
[0097]Any suitable means can be included in the present kits. For example, the means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein can comprise a peroxidase. Preferably, the chimeric protein and the peroxidase are formulated in a single composition.
[0098]Any suitable amadoriase, including the ones described in the above Sections B and C, can be used in the present kits. For example, the amadoriase can comprise a chimeric protein, which chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an amadoriase. Preferably, the chimeric protein comprises the amino acid sequence set forth in SEQ ID NO:5. Also preferably, the chimeric protein is encoded by the nucleotide sequence set forth in SEQ ID NO:6.
E. EXAMPLES
Example 1
Glycated Serum Protein Enzymatic Assay Kit
[0099]Intended Use. The exemplary assay kit is for determination of glycated serum proteins (fructosamine) in human serum. Fructosamine is formed due to a non-enzymatic Maillard reaction between glucose and amino acid residues of proteins. In diabetic patients, elevated blood glucose levels correlate with increased fructosamine formation. Fructosamine is a medium term indicator of diabetic control (2-3 weeks).
[0100]Assay Principle. The exemplary enzymatic assay for glycated serum proteins (GSP) uses Proteinase K to digest GSP into low molecular weight glycated protein fragments (GPF), and uses Diazyme's specific Fructosaminase®, a microorganism originated amadoriase to catalyze the oxidative degradation of Amadori product GPF to yield PF or amino acids, glucosone and H2O2. The H2O2 released is measured by a colorimetric Trinder end-point reaction. The absorbance at 550 nm is proportional to the concentration of glycated serum proteins (GSP).
TABLE-US-00007 TABLE 4 Reagent Table Reagent 1 (R1) Proteinase K, buffer Lyophilized 2 × 20 mL Reagent 2 (R2) Fructosaminase ®, HRP, buffer Lyophilized 2 × 5 mL
[0101]Test Samples. Use fresh patient serum or EDTA treated plasma samples. Plasma should be separated from cells immediately after collection. Samples can be stored at 4° C. for 2 weeks or up to 4 weeks when frozen.
[0102]Reconstitution. One vial of Reagents 1 is reconstituted with 20 mL of distilled water. Mix gently by inversion and then allow to stand for a minimum of 10 min at room temperature before use. The reconstituted R1 is stable for 4 weeks at 4° C. One vial of Reagent 2 is reconstituted with 5 mL of distilled water. Mix gently by inversion and then allow to stand at room temperature for minimum of 10 min before use. The reconstituted R2 is stable for 6 weeks at 4° C.
[0103]Assay Procedure
[0104]1. Pre-warm reconstituted R1 and R2 at room temperature.
[0105]2. Instrumental parameters. [0106]Wavelength: 550 nm; reference 700 nm [0107]Cuvette: 1 cm light path [0108]Temperature: 37° C.
[0109]3. Add 200 μL of the reconstituted R1 and 50 μL of sample or calibrator into cuvette. Mix and incubate for 5 min. Read absorbance at 550 nm as A1.
[0110]4. Add 50 μL of the reconstituted R2, mix and incubate for further 5 min and then read the absorbance at 550 nm as A2.
[0111]5. Reagent blank absorbance is read by using 50 μL of H2O instead of sample or calibrator.
[0112]Calculation
[0113]A=A2-A1
[0114]Concentration of glycated serum proteins (fructosamine) in sample:
Fructosamine ( mole / L ) = Δ Asample - Δ Ablank × Conc . of calibrator
[0115]Normal values. Adults (20-60 years) have a normal range of 122-285 μmol/L. Each laboratory should establish an expected range with a set of standards.
[0116]Linearity and Sensitivity. The assay is linear up to 1200 μmol/L and is sensitive at 30 μmol/L. The Diazyme Glycated Serum Protein (fructosamine) assay is precise with a mean inter-assay CV of <3% and mean intra assay CV of <2%. Assay data showed excellent correlation with the alternative fructosamine measurement method with r2=0.99.
[0117]Interferences. The following analyte concentrations were not found to affect the assay:
[0118]Ascorbic acid (4 mg/dL)
[0119]Bilirubin (2 mg/dL)
[0120]Glucose (1200 mg/dL
[0121]Hemoglobin (100 mg/dL)
[0122]Triglycerides (250 mg/dL)
[0123]Uric acid (15 mg/dL)
[0124]Calibration. Fructosamine Calibrator (Cat. No. DZ112A-S) is required for calibration.
[0125]Quality Control. Fructosamine Controls (low and high) (Cat. No. DZ112A-C1 and DZ112A-C3) are recommended to use as control sera. One control (low or high) should be tested after every 30 samples. Values should fall within a specific range. If these values fall outside the range and repetition excludes error, the following steps should be taken:
[0126]1. Check instrument settings and light source;
[0127]2. Check reaction temperature;
[0128]3. Check expiry date of kit and contents; and
[0129]4. Check the quality of the water used for reagents reconstitution.
REFERENCES
[0130]Armbuster D A, Fructosamine: Structure, Analysis and Clinical Usefulness. Clin. Chem. 1987; 33 (12): 2153-2163. [0131]Kouzuma, T. et al. An enzymatic method for the measurement of glycated albumin in biological samples. Clin. Chimi. Acta 2002; 324: 61-71.
Example 2
Glycated Serum Protein Assay Precision and Linearity
[0132]Method Comparison
[0133]Determined by running 2 replicates of a set of random samples using both Diazyme GSP kit and Randox Fructosamine kit in one run. The analytical performance characteristics determined by Diazyme GSP kit were comparable to those observed with Randox Fructosamine kit when assays were performed under the conditions as described in the Example 1 (See also FIG. 1).
[0134]Assay Linearity
[0135]Determined by running 2 replicates of a set of series diluted serum samples in one run. The assay is linear from 40-856 umole/L (See FIG. 2).
[0136]Interference
[0137]Determined by running 3 replicates each of a control sample in the absence and presence of various potential interference substances at indicated concentrations (See the following Table 5).
TABLE-US-00008 TABLE 5 Interference analysis Interfering substance Interfering substances concentration % Interference Ascorbic Acid 4 mg/dL -0.3 Bilirubin 2 mg/dL -0.6 Glucose 1200 mg/dL -0.6 Hemoglobin 100 mg/dL -4.4 Triglycerol 250 mg/dL 1.2 Uric Acid 15 mg/dL 5.3
Example 3
Assay for Glycated Hemoglobin HbA1c
[0138]A. Glycated Valine Measurement:
[0139]1. Mix 10 ul 170 mM Glycated Valine (G-Valine) (This value was assumed all valine was converted to G-Valine in the cooking procedure.) with 300 ul R1 (80 mMCHES, 30 mmMOPS, 0.9% BRIJ). This mixture serves as sample stock solution (GVR1).
[0140]2. Set spectrometer wavelength at 726 nm, temperature at 37° C. Pipette 150 ul R2 (30 mMMES, 1 mMCaCl2, 2 mMWST-3, 1570 U/ml Proteinase K) to a cuvette, add 0 ul, 2.5 ul, 5 ul, 10 ul, 15 ul, 20 ul above GVR1 respectively for dose response, make up the sample volume to 20 ul with H2O in R1, incubate for 5 min, get the first O.D. reading, then add 30 ul R3 (0.08 mMDA-64, 240 mMTris, 180 U/ml HRP, 20 U/ml FAOD), incubate for 3 min, get the second O.D. reading. Calculate the O.D. difference between these two readings, using 20 ul H2O in R1 as control. FIGS. 3 and 4 show the dose-dependent reaction with fructosyl-valine.
[0141]B. Glycated Hemoglobin Measurement:
[0142]1. Mix 10 ul high level Glycated Hemoglobin (G-hg(HHg)), 10 ul mid level G-hg(MHg), 10 ul normal level G-hg (NMHg) respectively with 300 ul R1.
[0143]2. Set spectrometer wavelength at 570 nm, temperature at 37° C. Pipette 150 ul R2 to cuvette, add 20 ul above Hg in R1 as sample, read the O.D. after 4 min incubation, using H2O in R1 as control. This O.D. reading gives the relative Hg concentration.
[0144]3. Change the spectrometer wavelength to 726 nm, get the first O.D. reading, add 30 ul R3, incubate for 5 min, get the second O.D. reading. Calculate the O.D. difference between these two readings. Normalize these O.D. differences of the three samples with their Hg concentrations. FIG. 5 shows the dose-dependent signal with patient hemoglobin digested with a proteinase (5 min. digestion).
[0145]The above examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Many variations to those described above are possible. Since modifications and variations to the examples described above will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.
Sequence CWU
1
23112PRTArtificial Sequence40%-100% identity to leader sequence 1Met Gly
Gly Ser Gly Asp Asp Asp Asp Leu Ala Leu 1 5
1026PRTArtificial SequenceFAD cofactor-binding consensus sequence 2Gly
Xaa Gly Xaa Xaa Gly 1 53437PRTArtificial Sequence40%-100%
identity to the amadoriase 3Ala Val Thr Lys Ser Ser Ser Leu Leu Ile Val
Gly Ala Gly Thr Trp 1 5 10
15Gly Thr Ser Thr Ala Leu His Leu Ala Arg Arg Gly Tyr Thr Asn Val
20 25 30Thr Val Leu Asp Pro Tyr Pro
Val Pro Ser Ala Ile Ser Ala Gly Asn 35 40
45Asp Val Asn Lys Val Ile Ser Ser Gly Gln Tyr Ser Asn Asn Lys
Asp 50 55 60Glu Ile Glu Val Asn Glu
Ile Leu Ala Glu Glu Ala Phe Asn Gly Trp65 70
75 80Lys Asn Asp Pro Leu Phe Lys Pro Tyr Tyr His
Asp Thr Gly Leu Leu 85 90
95Met Ser Ala Cys Ser Gln Glu Gly Leu Asp Arg Leu Gly Val Arg Val
100 105 110Arg Pro Gly Glu Asp Pro
Asn Leu Val Glu Leu Thr Arg Pro Glu Gln 115 120
125Phe Arg Lys Leu Ala Pro Glu Gly Val Leu Gln Gly Asp Phe
Pro Gly 130 135 140Trp Lys Gly Tyr Phe
Ala Arg Ser Gly Ala Gly Trp Ala His Ala Arg145 150
155 160Asn Ala Leu Val Ala Ala Ala Arg Glu Ala
Gln Arg Met Gly Val Lys 165 170
175Phe Val Thr Gly Thr Pro Gln Gly Arg Val Val Thr Leu Ile Phe Glu
180 185 190Asn Asn Asp Val Lys
Gly Ala Val Thr Gly Asp Gly Lys Ile Trp Arg 195
200 205Ala Glu Arg Thr Phe Leu Cys Ala Gly Ala Ser Ala
Gly Gln Phe Leu 210 215 220Asp Phe Lys
Asn Gln Leu Arg Pro Thr Ala Trp Thr Leu Val His Ile225
230 235 240Ala Leu Lys Pro Glu Glu Arg
Ala Leu Tyr Lys Asn Ile Pro Val Ile 245
250 255Phe Asn Ile Glu Arg Gly Phe Phe Phe Glu Pro Asp
Glu Glu Arg Gly 260 265 270Glu
Ile Lys Ile Cys Asp Glu His Pro Gly Tyr Thr Asn Met Val Gln 275
280 285Ser Ala Asp Gly Thr Met Met Ser Ile
Pro Phe Glu Lys Thr Gln Ile 290 295
300Pro Lys Glu Ala Glu Thr Arg Val Arg Ala Leu Leu Lys Glu Thr Met305
310 315 320Pro Gln Leu Ala
Asp Arg Pro Phe Ser Phe Ala Arg Ile Cys Trp Cys 325
330 335Ala Asp Thr Ala Asn Arg Glu Phe Leu Ile
Asp Arg His Pro Gln Tyr 340 345
350His Ser Leu Val Leu Gly Cys Gly Ala Ser Gly Arg Gly Phe Lys Tyr
355 360 365Leu Pro Ser Ile Gly Asn Leu
Ile Val Asp Ala Met Glu Gly Lys Val 370 375
380Pro Gln Lys Ile His Glu Leu Ile Lys Trp Asn Pro Asp Ile Ala
Ala385 390 395 400Asn Arg
Asn Trp Arg Asp Thr Leu Gly Arg Phe Gly Gly Pro Asn Arg
405 410 415Val Met Asp Phe His Asp Val
Lys Glu Trp Thr Asn Val Gln Tyr Arg 420 425
430Asp Ile Ser Lys Leu 435417PRTArtificial
Sequence40%-100% identity of the second bacterial leader sequence
4Lys Gly Glu Leu Glu Gly Leu Pro Ile Pro Asn Pro Leu Leu Arg Thr 1
5 10 15Gly5472PRTArtificial
Sequencechimeric protein 5Met Gly Gly Ser Gly Asp Asp Asp Asp Leu Ala Leu
Ala Val Thr Lys 1 5 10
15Ser Ser Ser Leu Leu Ile Val Gly Ala Gly Thr Trp Gly Thr Ser Thr
20 25 30Ala Leu His Leu Ala Arg Arg
Gly Tyr Thr Asn Val Thr Val Leu Asp 35 40
45Pro Tyr Pro Val Pro Ser Ala Ile Ser Ala Gly Asn Asp Val Asn
Lys 50 55 60Val Ile Ser Ser Gly Gln
Tyr Ser Asn Asn Lys Asp Glu Ile Glu Val65 70
75 80Asn Glu Ile Leu Ala Glu Glu Ala Phe Asn Gly
Trp Lys Asn Asp Pro 85 90
95Leu Phe Lys Pro Tyr Tyr His Asp Thr Gly Leu Leu Met Ser Ala Cys
100 105 110Ser Gln Glu Gly Leu Asp
Arg Leu Gly Val Arg Val Arg Pro Gly Glu 115 120
125Asp Pro Asn Leu Val Glu Leu Thr Arg Pro Glu Gln Phe Arg
Lys Leu 130 135 140Ala Pro Glu Gly Val
Leu Gln Gly Asp Phe Pro Gly Trp Lys Gly Tyr145 150
155 160Phe Ala Arg Ser Gly Ala Gly Trp Ala His
Ala Arg Asn Ala Leu Val 165 170
175Ala Ala Ala Arg Glu Ala Gln Arg Met Gly Val Lys Phe Val Thr Gly
180 185 190Thr Pro Gln Gly Arg
Val Val Thr Leu Ile Phe Glu Asn Asn Asp Val 195
200 205Lys Gly Ala Val Thr Gly Asp Gly Lys Ile Trp Arg
Ala Glu Arg Thr 210 215 220Phe Leu Cys
Ala Gly Ala Ser Ala Gly Gln Phe Leu Asp Phe Lys Asn225
230 235 240Gln Leu Arg Pro Thr Ala Trp
Thr Leu Val His Ile Ala Leu Lys Pro 245
250 255Glu Glu Arg Ala Leu Tyr Lys Asn Ile Pro Val Ile
Phe Asn Ile Glu 260 265 270Arg
Gly Phe Phe Phe Glu Pro Asp Glu Glu Arg Gly Glu Ile Lys Ile 275
280 285Cys Asp Glu His Pro Gly Tyr Thr Asn
Met Val Gln Ser Ala Asp Gly 290 295
300Thr Met Met Ser Ile Pro Phe Glu Lys Thr Gln Ile Pro Lys Glu Ala305
310 315 320Glu Thr Arg Val
Arg Ala Leu Leu Lys Glu Thr Met Pro Gln Leu Ala 325
330 335Asp Arg Pro Phe Ser Phe Ala Arg Ile Cys
Trp Cys Ala Asp Thr Ala 340 345
350Asn Arg Glu Phe Leu Ile Asp Arg His Pro Gln Tyr His Ser Leu Val
355 360 365Leu Gly Cys Gly Ala Ser Gly
Arg Gly Phe Lys Tyr Leu Pro Ser Ile 370 375
380Gly Asn Leu Ile Val Asp Ala Met Glu Gly Lys Val Pro Gln Lys
Ile385 390 395 400His Glu
Leu Ile Lys Trp Asn Pro Asp Ile Ala Ala Asn Arg Asn Trp
405 410 415Arg Asp Thr Leu Gly Arg Phe
Gly Gly Pro Asn Arg Val Met Asp Phe 420 425
430His Asp Val Lys Glu Trp Thr Asn Val Gln Tyr Arg Asp Ile
Ser Lys 435 440 445Leu Lys Gly Glu
Leu Glu Gly Leu Pro Ile Pro Asn Pro Leu Leu Arg 450
455 460Thr Gly His His His His His His465
47061419DNAArtificial Sequencenucleotide sequence encoding a chimeric
protein 6atgggaggtt cgggtgacga tgatgacctg gctctcgccg tcactaagtc
atcatctctc 60ctgatcgttg gtgccgggac ttggggcacc tcaacggctc tgcacctcgc
gcgccgcgga 120tataccaacg ttaccgtgct ggacccctat cctgtcccta gcgccatctc
cgccggaaac 180gacgtgaaca aagtcattag cagtggccaa tattcgaata acaaagacga
aatcgaagtg 240aatgagatct tggcggaaga ggcgtttaac ggttggaaga acgacccgct
tttcaaaccg 300tattatcatg atacgggcct gctgatgtct gcttgctcgc aggagggcct
ggatcgcctg 360ggcgtccggg tacgtccggg cgaggatcct aatctggtgg aacttacccg
cccggagcaa 420tttcgtaaac tggccccgga aggcgtgttg caaggtgatt ttccgggttg
gaaagggtac 480tttgcgcgtt ccggcgctgg ctgggcacat gcaaggaatg ccttagtggc
agcagcacgc 540gaagcacagc gcatgggtgt aaaatttgtt actggcaccc cgcagggtcg
tgtagtcacg 600ttaatctttg aaaataacga tgtaaaaggt gccgttacgg gcgatggcaa
aatttggaga 660gcggaacgta cattcctgtg tgctggggct agcgcgggtc agttcctaga
tttcaagaat 720caacttcgac caaccgcttg gaccctggta cacattgcgt taaaaccgga
agaacgtgcg 780ttgtacaaaa atataccggt tatctttaac atcgaacggg ggtttttctt
tgaacccgat 840gaggagcgcg gtgagattaa aatatgcgat gaacacccgg gctacacaaa
tatggtccag 900agtgcagacg gcacgatgat gagcattccg ttcgaaaaaa cccagattcc
aaaagaagcc 960gaaacgcgcg ttcgggccct gctgaaagag acaatgcccc agctggcaga
ccgtccattc 1020agcttcgcac gcatttgctg gtgtgccgat accgcgaatc gcgaattcct
gatagatcga 1080catccgcagt accacagtct tgtgttgggc tgtggtgcga gcggaagagg
gtttaaatat 1140ctgccttcta ttgggaatct cattgttgac gcgatggaag gtaaagtgcc
gcaaaaaatt 1200cacgaattaa tcaagtggaa cccggacatt gcggcgaacc gtaactggcg
tgatactctg 1260gggcgttttg gcggtccaaa tcgtgtgatg gattttcatg atgtgaagga
atggaccaat 1320gttcagtatc gtgatatttc caagctgaaa ggagagttgg aaggtaagcc
aatccctaac 1380ccgttactgc gcacaggcca tcaccatcat catcattaa
1419739PRTArtificial Sequencesequence homology between the
N-terminal sequence of Amadoriases Ia 7Ala Pro Ser Ile Leu Ser Thr
Glu Ser Ser Ile Xaa Val Ile Gly Ala 1 5 10
15Gly Thr Trp Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 20 25 30Gly Gly
Gly Gly Gly Gly Gly 35839PRTArtificial SequenceSequence homology
between the N-terminal sequence of Amadoriase Ib 8Ala Pro Ser Ile
Leu Ser Thr Glu Ser Ser Ile Ile Val Ile Gly Ala 1 5
10 15Gly Thr Trp Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 20 25
30Gly Gly Gly Gly Gly Gly Gly 35939PRTArtificial SequenceSequence
homology between the N-terminal sequence of Amadoriase Ic 9Ser Thr
Glu Ser Ser Ile Ile Val Ile Gly Ala Gly Thr Trp Gly Cys 1 5
10 15Ser Thr Ala Leu Leu Leu Leu Leu
Leu Leu Leu Leu Leu Leu Leu Leu 20 25
30Leu Leu Leu Leu Leu Leu Leu 351039PRTArtificial
SequenceSequence homology between the N-terminal sequence of
Amadoriase II 10Ala Val Thr Lys Ser Ser Ser Leu Leu Ile Val Gly Ala Gly
Thr Trp 1 5 10 15Gly Thr
Ser Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr 20
25 30Thr Thr Thr Thr Thr Thr Thr
35117PRTArtificial Sequenceexemplary epitope tag 11Asp Tyr Lys Asp Asp
Asp Lys 1 5129PRTArtificial Sequenceexemplary epitope tag
12Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 51311PRTArtificial
Sequenceexemplary epitope tag 13Cys Gln Asp Leu Pro Gly Asn Asp Asn Ser
Thr 1 5 101410PRTArtificial
Sequenceexemplary epitope tag 14Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1
5 10156PRTArtificial Sequenceexemplary
epitope tag 15His His His His His His 1 5166PRTArtificial
Sequenceexemplary epitope tag 16Asp Thr Tyr Arg Tyr Ile 1
5176PRTArtificial Sequenceexemplary epitope tag 17Glu Tyr Met Pro Met Glu
1 51811PRTArtificial Sequenceexemplary epitope tag 18Ala
Ser Met Thr Gly Gly Gln Gln Met Gly Arg 1 5
101910PRTArtificial Sequenceexemplary epitope tag 19Ser Phe Pro Gln Phe
Lys Pro Gln Glu Ile 1 5
102012PRTArtificial Sequenceexemplary epitope tag 20Lys Gly Phe Ser Tyr
Phe Gly Glu Asp Leu Met Pro 1 5
10216PRTArtificial Sequenceexemplary epitope tag 21Gln Tyr Pro Ala Leu
Thr 1 52211PRTArtificial Sequenceexemplary epitope tag
22Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly Asp 1 5
102310PRTArtificial Sequenceexemplary epitope tag 23Glu Val His
Thr Asn Gln Asp Pro Leu Asp 1 5 10
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