WO2010041715A1 - 新規フルクトシルペプチドオキシダーゼ - Google Patents
新規フルクトシルペプチドオキシダーゼ Download PDFInfo
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- WO2010041715A1 WO2010041715A1 PCT/JP2009/067565 JP2009067565W WO2010041715A1 WO 2010041715 A1 WO2010041715 A1 WO 2010041715A1 JP 2009067565 W JP2009067565 W JP 2009067565W WO 2010041715 A1 WO2010041715 A1 WO 2010041715A1
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
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- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/9065—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5)
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Definitions
- the present invention relates to a protein having novel fructosyl peptide oxidase activity, DNA encoding the protein, a method for producing the protein, a method for measuring a glycated protein using the protein, and a reagent for measuring a glycated protein containing the protein.
- Glycated protein is contained in body fluids such as blood and biological samples such as hair.
- concentration of glycated protein present in blood depends on the concentration of saccharides such as glucose dissolved in serum.
- hemoglobin A1c which is a glycated protein in blood
- HPLC high performance liquid chromatography
- an immunoassay method using an antigen-antibody reaction for example, Non-Patent Document 3
- Enzymatic measurement methods have been developed. For example, a method using a protease and fructosyl peptide oxidase (Patent Document 1) has been developed. Enzymatic measurement methods can be applied to versatile automatic analyzers, and their operation is simple.
- the fructosyl peptide oxidase used in the enzymatic measurement method has a CN bond in the ketose derivative produced by the Amadori rearrangement of glucosylamine produced by the reaction of hemiacetal of glucose and the N-terminal amino group of the peptide, It is an enzyme that catalyzes a reaction that is oxidatively cleaved in the presence of oxygen molecules to produce sugar oxone ( ⁇ -ketoaldehyde form), peptide, and hydrogen peroxide.
- ⁇ -FVH ⁇ -fructosylvalylhistidine
- a fructosyl peptide oxidase derived from the genus Acaetomiera, the genus Caetomium (patent document 3), the genus Carbaria (patent document 2), the rose family, the grape family, the seriaceae (patent document 4), the ginger family (patent document 5) are known.
- the fructosyl peptide oxidase reported so far is (1)
- the activity against ⁇ -glycated dipeptide ( ⁇ -fructosylvalylhistidine) is not necessarily high compared to ⁇ -glycated amino acid (for example, ⁇ -fructosyl valine), (2)
- ⁇ -glycated amino acid for example, ⁇ -fructosyl valine
- it in addition to the N-terminal ⁇ -glycated dipeptide, it also acts on ⁇ -glycated amino acid ( ⁇ -fructosyl lysine) in which saccharide is bound to the ⁇ -amino group of lysine, and in hemoglobin A1c measurement Increasing the measured value
- (3) In the case of a measurement method using an enzyme, the enzyme becomes unstable during measurement or storage. There were disadvantages such as.
- Patent Document 4 an enzyme having reduced reactivity to ⁇ -fructosyl lysine by artificially introducing a mutation into fructosyl peptide oxidase
- Patent Document 4 Increased enzymes
- Non-patent Document 4 the existence of an enzyme that has overcome the above disadvantages (1) to (3) at a high level is not yet known.
- the present invention has been made in view of the above problems, and its object is to have high specificity to ⁇ -glycated dipeptide ( ⁇ -fructosyl valylhistidine) and to have excellent stability of fructosyl peptide oxidase activity. It is to provide protein. Another object of the present invention is to provide a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, a fructose using the transformant and the like.
- An object of the present invention is to provide a method for producing a protein having tosyl peptide oxidase activity, a method for measuring a glycated protein using the protein, and a reagent for measuring a glycated protein containing the protein.
- the present invention relates to the following (1) to (17).
- the protein according to any one of [1] to [4] below.
- [1] A protein having the amino acid sequence represented by SEQ ID NO: 1
- a protein having oxidase activity [3]
- a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having fructosyl peptide oxidase activity [4] Deposit No.
- FERM BP-11026 A protein having fructosyl peptide oxidase activity encoded by an expression plasmid carried by the deposited Escherichia coli XL1-Blue MRF 'strain (2) The protein according to (1), comprising the amino acid sequence represented by SEQ ID NO: 3. (3) The DNA according to any one of [1] to [3] below.
- DNA encoding the protein according to (1) [2] DNA having the base sequence represented by SEQ ID NO: 2 [3] DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 and encodes a protein having fructosyl peptide oxidase activity (4) The DNA according to (3), which encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 3. (5) The DNA according to (3), comprising the base sequence represented by SEQ ID NO: 4. (6) A recombinant DNA containing the DNA according to any one of (3) to (5). (7) A transformant having the recombinant DNA according to (6).
- the transformant according to (7) is cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture.
- a sample is reacted with a proteolytic enzyme to produce a glycated peptide, then the produced glycated peptide is reacted with the protein described in (1) or (2), and produced by a reaction between the glycated peptide and the protein.
- a method for measuring a glycated protein in a sample comprising measuring a substance consumed or a substance consumed in a reaction between the glycated peptide and the protein.
- the measuring method according to (9), wherein the glycated protein is glycated hemoglobin.
- (11) The measuring method according to (10), wherein the glycated hemoglobin is hemoglobin A1c.
- a reagent for measuring a glycated protein comprising a proteolytic enzyme and the protein according to (1) or (2).
- the reagent according to (13), wherein the product is hydrogen peroxide.
- a highly specific and stable fructosyl peptide oxidase activity protein for ⁇ -glycated dipeptide ( ⁇ -fructosyl valylhistidine), a DNA encoding the protein, a method for producing the protein,
- a method for measuring a glycated protein using the protein and a reagent for measuring a glycated protein containing the protein are provided.
- the amino acid circled above represents the mutation site from the fructosyl peptide oxidase-producing bacterium (gene source), and the underlined amino acid represents the mutation site relative to the amino acid sequence of FPOX-9. It is a figure which shows the DNA sequence of FPOX-15. The bases marked with circles indicate the mutation sites in the DNA sequence of the fructosyl peptide oxidase-producing bacterium (gene source), and the underlined bases indicate the mutation sites in the DNA sequence of FPOX-9. To express.
- Protein of the present invention As the protein of the present invention, [1] a protein having the amino acid sequence represented by SEQ ID NO: 1, [2] a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having fructosyl peptide oxidase activity; [3] a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having fructosyl peptide oxidase activity; [4] A protein having fructosyl peptide oxidase activity encoded by an expression plasmid carried by the Escherichia coli XL1-Blue MRF ′ strain deposited under the deposit number FERM BP-11026.
- a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added and having fructosyl peptide oxidase activity is Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). ) (Hereinafter abbreviated as Molecular Cloning 2nd edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research , 10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl.
- site-directed mutagenesis is introduced into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. To get by It can be.
- the number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a known method such as the above-described site-directed mutagenesis method, 1 to several tens, The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
- amino acids When one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids are deleted, substituted or added at any position in the same sequence. Also good.
- amino acid positions at which amino acid deletion or addition can be performed include one to several amino acids on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 1.
- Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
- amino acids included in the same group can be substituted for each other.
- Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
- Group B aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
- Group C asparagine, glutamine
- D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
- Group E proline, 3 -Hydroxyproline, 4-hydroxyproline
- Group F serine, threonine, homoserine
- Group G phenyl, threonine
- the homology with the amino acid sequence represented by SEQ ID NO: 1 is 80% or more, preferably 90% or more, more preferably 94% or more, It is desirable that the homology is 98% or more, particularly preferably 99% or more.
- a protein having fructosyl peptide oxidase activity is also a protein of the present invention.
- protein of the present invention for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 3 can be shown.
- the protein of the present invention is a protein having fructosyl peptide oxidase activity
- a transformant expressing the protein of the present invention is prepared using a DNA recombination method, and the transformant is used. After the production of the protein of the present invention using ⁇ -FVH as a substrate, hydrogen peroxide produced by reaction with the substrate can be measured.
- the protein of the present invention has the following properties.
- Action Oxidizes glycated peptide using molecular oxygen to produce sugar oxone ( ⁇ -ketoaldehyde), peptide and hydrogen peroxide.
- Substrate specificity high reactivity with ⁇ -FVH and low reactivity with ⁇ -fructosyl lysine (hereinafter abbreviated as ⁇ -FK).
- the protein of the present invention has high activity for ⁇ -FVH and low reactivity for ⁇ -FK is, for example, that ⁇ -FVH and ⁇ -FK are used as substrates and ⁇ - This can be confirmed by measuring the activity ratio of FVH ( ⁇ -FVH / ⁇ -FK).
- the optimum pH and stable pH range of the fructosyl peptide oxidase activity of the protein of the present invention is not particularly limited, but the optimum pH is preferably around 6.0 to 7.0, and the stable pH is treated at 40 ° C. for 10 minutes.
- a pH of 6.0 to 9.0 is preferred.
- fructosyl peptide oxidase The activity of fructosyl peptide oxidase is measured by the following method, and the amount of enzyme that produces 1 ⁇ mol of hydrogen peroxide per minute from ⁇ -FVH is defined as 1 unit (U).
- Solution A Coloring solution
- Solution A-1 4-Aminoantipyrine is dissolved in ion-exchanged water so as to have a concentration of 2.4 mmol / L.
- Solution A-2 N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine (EMSE) is dissolved in ion-exchanged water to a concentration of 32 mmol / L.
- Solution B Peroxidase solution Peroxidase (110 U / mg, manufactured by Toyobo Co., Ltd.) is dissolved in 0.1 mol / L phosphate buffer (pH 7.0) to a concentration of 2 mg / mL.
- Solution C Substrate solution ⁇ -FVH or ⁇ -FK (manufactured by Peptide Institute) is dissolved in 0.1 mol / L phosphate buffer (pH 7.0) to a concentration of 10 mmol / L.
- Escherichia coli XL1-Blue MRF '/ pTrcFPOX-9 carrying a plasmid (expression plasmid) that expresses a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is an independent administrative agency. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center. The contents specifying the deposit are described below.
- the E. coli XL1-Blue® MRF ′ strain deposited under the deposit number FERM®BP-11026, an expression plasmid carried by the strain, and a protein having fructosyl peptide oxidase activity encoded by the plasmid are also included in the present invention.
- DNA of the present invention As the DNA of the present invention, [1] DNA encoding the protein of the present invention of [1] to [3] in 1 above, [2] hybridizes under stringent conditions with DNA having the base sequence represented by SEQ ID NO: 2 and [3] DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2; DNA encoding a protein having fructosyl peptide oxidase activity, Can be mentioned.
- hybridize means that the DNA of interest has hybridized to DNA having a specific base sequence or a part of the DNA. Accordingly, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or DNA of a length that can be used as an oligonucleotide primer for PCR analysis. May be.
- DNA used as a probe include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases, preferably 15 bases or more. .
- the above stringent conditions include, for example, a filter in which DNA is immobilized and a probe DNA, 50% formamide, 5 ⁇ SSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / After incubation overnight at 42 ° C. in a solution containing L sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g / L denatured salmon sperm DNA, for example, about
- the conditions for washing the filter in a 0.2 ⁇ SSC solution at 65 ° C. can be mentioned, but lower stringent conditions can also be used.
- Stringent conditions can be changed by adjusting the concentration of formamide (the lower the formamide concentration, the lower the stringency), and changing the salt concentration and temperature conditions.
- 6 ⁇ SSCE (20 ⁇ SSCE is 320mol / L sodium chloride, 0.2 mol / L sodium dihydrogen phosphate, 0.02 mol / L EDTA, pH 7.4), 0.5%
- Conditions of washing with 1x SSC, 0.1% SDS solution at 50 ° C after overnight incubation at 37 ° C in a solution containing 1% SDS, 30% formamide, 100 ⁇ g / L denatured salmon sperm DNA Can be mentioned.
- examples of lower stringent conditions include conditions in which hybridization is performed using a solution having a high salt concentration (for example, 5 ⁇ SSC) under the low stringent conditions described above, and then washed.
- the various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of the hybridization experiment.
- the addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.
- the DNA that can hybridize under the above stringent conditions includes, for example, at least the base sequence represented by SEQ ID NO: 2 when calculated based on the above parameters using a program such as BLAST and FASTA described above. Examples thereof include DNA comprising a base sequence having a homology of 80% or more, preferably 90% or more, more preferably 94% or more, further preferably 98% or more, and particularly preferably 99% or more.
- the DNA that hybridizes with the above DNA under stringent conditions is a DNA that encodes a protein having fructosyl peptide oxidase activity.
- a recombinant DNA that expresses the DNA is prepared, and the recombinant The protein is purified from a culture obtained by culturing a microorganism obtained by introducing DNA into a host cell, the purified protein is used as an enzyme source, ⁇ -FVH is used as a substrate, and reaction with the substrate is performed. This can be done by measuring the hydrogen peroxide produced.
- Examples of the DNA of the present invention include DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, DNA consisting of the base sequence represented by SEQ ID NO: 4, and the like.
- Transformant of the present invention a transformant obtained by transforming a host cell by a known method using a recombinant DNA containing the above-mentioned DNA can be mentioned,
- host cells include bacteria, yeasts, animal cells, insect cells and plant cells, preferably bacteria, more preferably prokaryotic cells, and more preferably microorganisms belonging to the genus Escherichia .
- the DNA of the present invention belongs to microorganisms such as filamentous fungi, preferably belonging to the genus Aspergillus , Emericella , etc., using, for example, a probe that can be designed based on the nucleotide sequence represented by SEQ ID NO: 2. It can be obtained by microorganisms, particularly preferably microorganisms belonging to Emericella nidulans and the like .
- a sequence having a homology of 99% or more is searched, and based on the base sequence obtained by the search, the method of the present invention is carried out by the method described above from the chromosomal DNA, cDNA library, etc. of the organism having the base sequence. DNA or DNA used in the production method of the present invention can also be obtained.
- the obtained DNA is cut as it is or with an appropriate restriction enzyme and incorporated into a vector by a conventional method.
- a commonly used nucleotide sequence analysis method such as the dideoxy method [ Proc. Natl. Acad. Sci., USA, 74 , 5463 (1977)] or 373A DNA sequencer (manufactured by Perkin Elmer) or the like to analyze the base sequence of the DNA. Can be determined.
- pBluescriptII KS (+) (Stratagene)
- pDIRECT [Nucleic Acids Res., 18 , 6069 (1990)]
- pCR-Script Amp SK (+) (Stratagene)
- PT7Blue manufactured by Novagen
- pCR II manufactured by Invitrogen
- pCR-TRAP manufactured by Gene Hunter
- Examples of host cells include microorganisms belonging to the genus Escherichia.
- Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli W1485, Escherichia coli. ⁇ Cori JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415, etc. it can.
- any method can be used as long as it is a method for introducing DNA into the host cell.
- a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)]
- protoplast method Japanese Patent Laid-Open No. 63-248394
- electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.
- the full-length DNA can be obtained by Southern hybridization to a chromosomal DNA library using the partial length DNA as a probe.
- the target DNA can be prepared by chemical synthesis using a 8905 type DNA synthesizer manufactured by Perceptive Biosystems. Examples of the DNA obtained as described above include a DNA having the base sequence represented by SEQ ID NO: 2.
- Method for producing transformant used in production method of the present invention Based on the DNA of the present invention, a DNA fragment having an appropriate length containing a portion encoding the protein of the present invention is prepared as necessary. In addition, a transformant with an improved production rate of the protein can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for host expression.
- a recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.
- a transformant producing the protein of the present invention can be obtained by introducing the recombinant DNA into a host cell suitable for the expression vector. Any host cell can be used as long as it can express the gene of interest, such as bacteria, yeast, animal cells, insect cells, and plant cells.
- the expression vector one that can autonomously replicate in the host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.
- the recombinant DNA having the DNA of the present invention can autonomously replicate in the prokaryote, and at the same time, a promoter, a ribosome binding sequence, the DNA of the present invention, a transcription termination It is preferably a recombinant DNA composed of a sequence. A gene that controls the promoter may also be included.
- Expression vectors include pColdI (Takara Bio), pCDF-1b, pRSF-1b (Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Bio) Science), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), pET-3 (Novagen), pKYP10 58-110600), pKYP200 [Agric. Biol. Chem., 48 , 669 (1984)], pLSA1 [Agric. Biol. Chem., 53 , 277 (1989)], pGEL1 [Proc.
- any promoter that functions in a host cell such as Escherichia coli may be used.
- trp promoter P trp
- P lac cited lac promoter
- P L promoter P L promoter
- P R promoter P SE promoter
- promoters derived from Escherichia coli or phage such as, SPO1 promoter, SPO2 promoter, the penP promoter and the like be able to.
- artificially designed and modified promoters such as a promoter in which two P trp are connected in series, tac promoter, lacT7 promoter, let I promoter, and the like can also be used.
- xylA promoter (Appl. Microbiol. Biotechnol., 35 , 594-599 (1991)] for expression in microorganisms belonging to the genus Bacillus and P54- for expression in microorganisms belonging to the genus Corynebacterium 6 Promoters [Appl. Microbiol. Biotechnol., 53 , 674-679 (2000)] can also be used.
- a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
- a transcription termination sequence is not necessarily required, but it is preferable to place the transcription termination sequence immediately below the structural gene. Examples of such recombinant DNA include pET21-plu1440.
- any method can be used as long as it is a method for introducing DNA into the host cell.
- a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)]
- protoplast method Japanese Patent Laid-Open No. 63-248394
- electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.
- yeast strains When yeast strains are used as host cells, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like can be used as expression vectors.
- Any promoter may be used as long as it functions in yeast strains.
- PHO5 promoter PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter And promoters such as MF ⁇ 1 promoter and CUP 1 promoter.
- Saccharomyces (Saccharomyces) genus Schizosaccharomyces (Schizosaccharomyces) genus Kluyveromyces (Kluyveromyces) genus Trichosporon (Trichosporon) genus Shiwaniomaisesu (Schwanniomyces) genus Pichia (Pichia) sp or Candida, ( Yeast strains belonging to the genus Candida, etc., specifically, Saccharomyces cerevisiae , Schizosaccharomyces pombe , Kluyveromyces lactis , Trichosporon pullulans (Trichosporon pullulans), Shiwaniomaisesu-Arubiusu (Schwanniomyces alluvius), Pichia pastoris (Pichia pastoris), can be mentioned Candida utilis (Candida utilis), and the like.
- any method for introducing DNA into yeast can be used.
- electroporation Metals Enzymol., 194 , 182 (1990)
- ferroplast method Proc. Natl. Acad. Sci., USA, 81 , 4889 (1984)
- lithium acetate method J. Bacteriol., 153 , 163 (1983)
- examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 (Japanese Patent Laid-Open No. 3-22979), pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329 , 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem, 101 , 1307 (1987)], pAGE210, pAMo, pAMoA and the like can be used.
- CMV cytomegalovirus
- SV40 early promoter or metallothionein promoter SV40 early promoter or metallothionein promoter
- retrovirus Promoter heat shock promoter
- SR ⁇ promoter SR ⁇ promoter
- an IE gene enhancer of human CMV may be used together with a promoter.
- Host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, human cells such as Namalwa cells or Namalva KJM-1 cells, human fetal kidney cells, human leukemia cells, African green monkey kidney cells CHO cells that are Chinese hamster cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and the like.
- mice myeloma cells As mouse myeloma cells, SP2 / 0, NSO, etc., as rat myeloma cells, YB2 / 0, etc., as human fetal kidney cells, HEK293 (ATCC CRL-1573), as human leukemia cells, as BALL-1, etc., Africa Examples of green monkey kidney cells include COS-1 and COS-7.
- any method for introducing DNA into animal cells can be used.
- electroporation [Cytotechnology, 3 , 133 (1990)]
- calcium phosphate examples include the method described in JP-A-2-27075, the lipofection method [Proc. Natl. Acad. Sci., USA, 84 , 7413 (1987)], Virology, 52 , 456 (1973).
- Baculovirus Expression Vectors A Laboratory Manual, WH Freeman and Company, New York (1992), Current Protocols in Molecular Biology, Molecular Biology, A Laboratory Manual, Bio Proteins can be produced by the method described in / Technology, 6 , 47 (1988). That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the insect cells are further infected with the recombinant virus to produce a protein. it can.
- Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
- the baculovirus for example, an autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
- insect cells podocytes of Spodoptera frugiperda , ovary cells of Trichoplusia ni , cultured cells derived from silkworm ovary, and the like can be used.
- Spodoptera frugiperda ovary cells are Sf9, Sf21 (Baculovirus Expression Vectors A Laboratory Manual), etc., Trichopulcia ni ovary cells are High 5, BTI-TN-5B1-4 (manufactured by Invitrogen) Examples of cultured cells derived from silkworm ovary include Bombyx mori N4.
- Examples of the method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-27075), the lipofection method [Proc. Natl. Acad Sci., USA, 84 , 7413 (1987)].
- examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter may be used as long as it functions in plant cells. Examples thereof include cauliflower mosaic virus (CaMV) 35S promoter and rice actin 1 promoter. Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.
- a method for introducing a recombinant vector into a plant cell any method can be used as long as it is a method for introducing DNA into a plant cell. For example, a method using Agrobacterium (Japanese Patent Laid-Open No.
- JP-A-60-251887 a method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813) and the like.
- the transformant obtained by the above method 5 is cultured in a medium, the protein of the present invention is produced and accumulated in the culture, and the protein is produced by collecting from the culture. can do.
- the host of the transformant for producing the protein of the present invention may be any of bacteria, yeast, animal cells, insect cells, plant cells, etc., preferably bacteria, more preferably belonging to the genus Escherichia. And microorganisms belonging to Escherichia coli, more preferably. When expressed in yeast, animal cells, insect cells, or plant cells, a protein with a sugar or sugar chain added can be obtained.
- the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
- the medium As a medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host, the medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism, Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.
- a prokaryote such as Escherichia coli or a eukaryote such as yeast
- the medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism
- Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.
- the carbon source may be anything that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol, Alcohols such as propanol can be used.
- Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digests thereof, and the like can be used.
- monopotassium phosphate dipotassium phosphate
- magnesium phosphate magnesium sulfate
- sodium chloride ferrous sulfate
- manganese sulfate copper sulfate
- calcium carbonate calcium carbonate
- the culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
- the culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 to 7 days.
- the pH is maintained at 3.0 to 9.0.
- the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
- antibiotics such as an ampicillin and a tetracycline, to a culture medium as needed during culture
- an inducer may be added to the medium as necessary.
- an inducer For example, isopropyl ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic acid is used when culturing a microorganism transformed with an expression vector using the trp promoter. Etc. may be added to the medium.
- a commonly used RPMI1640 medium J. Am. Med. Assoc., 199 , 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], DMEM medium [Virology, 8 , 396 (1959)], 199 medium [Proc. Soc. Biol. Med., 73 , 1 (1950)] or fetal calf serum in these media Etc.
- the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and the presence of 5% CO 2 .
- TNM-FH medium manufactured by Farmingen
- Sf-900 II SFM medium manufactured by Life Technologies
- ExCell400 ExCell405 all manufactured by JRH Biosciences
- Grace's Insect Medium [Nature, 195 , 788 (1962)] and the like
- the culture is usually carried out for 1 to 5 days under conditions of pH 6 to 7, 25 to 30 ° C.
- antibiotics such as a gentamicin
- a transformant obtained using a plant cell as a host can be cultured as a cell or differentiated into a plant cell or organ.
- a medium for cultivating the transformant a generally used Murashige and Skoog (MS) medium, a White medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these mediums or the like is used. be able to.
- the culture is usually carried out under conditions of pH 5-9 and 20-40 ° C. for 3-60 days.
- Examples of the method for producing the protein of the present invention include a method for producing in the host cell, a method for secreting it outside the host cell, and a method for producing it on the outer cell membrane of the host cell.
- the structure can be changed.
- a protein containing the active site of the protein of the present invention can be produced in a form in which a signal peptide is added before the protein to actively secrete the protein outside the host cell. it can.
- the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
- the protein of the invention can also be produced.
- the transformant producing the protein of the present invention is an animal individual or a plant individual
- the protein is bred or cultivated according to a normal method, the protein is produced and accumulated, and the protein is collected from the animal individual or plant individual.
- the protein can be produced.
- a method for producing the protein of the present invention using an animal individual for example, a known method [Am. J. Clin. Nutr., 63 , 639S (1996), Am. J. Clin. Nutr., 63 , 627S ( 1996), Bio / Technology, 9 , 830 (1991)], a method for producing the protein of the present invention in an animal constructed by introducing a gene.
- a transgenic non-human animal into which the DNA of the present invention or the DNA used in the production method of the present invention is introduced is bred, and the protein of the present invention is produced and accumulated in the animal. By collecting the protein from the inside, the protein can be produced.
- Examples of the place where the protein in the animal is produced and accumulated include milk of the animal (Japanese Patent Laid-Open No. 63-309192) and eggs.
- Any promoter can be used as long as it functions in animals.
- a casein promoter, ⁇ casein promoter, ⁇ lactoglobulin promoter, whey acidity, which is a mammary cell specific promoter can be used.
- a protein promoter or the like is preferably used.
- a transgenic plant introduced with a DNA encoding the protein of the present invention can be produced by a known method [tissue culture, 20 (1994), tissue culture, 21 (1995 ), Trends Biotechnol., 15 , 45 (1997)], producing and accumulating the protein in the plant, and collecting the protein from the plant to produce the protein. can give.
- a usual enzyme isolation and purification method can be used.
- the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton. Cells are disrupted with a Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
- an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), a cation using a resin such as S-Sepharose FF (manufactured by GE Healthcare Bioscience) Exchange chromatography method, hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. These methods can be used alone or in combination to obtain a purified preparation.
- a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75
- the protein when the protein is produced by forming an insoluble substance in the cell, the protein is similarly collected from the precipitate fraction obtained by crushing and then centrifuging the cell, and the protein is obtained by a conventional method. After recovery, the protein insoluble material is solubilized with a protein denaturant.
- the solubilized solution is diluted or dialyzed into a dilute solution that does not contain a protein denaturing agent or the concentration of the protein denaturing agent does not denature the protein, and the protein is constituted into a normal three-dimensional structure.
- a purified sample can be obtained by the same isolation and purification method.
- the protein of the present invention or a derivative such as a sugar modification product thereof is secreted extracellularly
- the protein or its derivative such as a sugar adduct can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
- the protein thus obtained include a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
- the protein of the present invention can be produced as a fusion protein with another protein, and purified using affinity chromatography using a substance having affinity for the fused protein.
- affinity chromatography using a substance having affinity for the fused protein.
- the method described in Law et al. Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)] JP-A-5-336963, WO94 / 23021
- the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.
- the protein of the present invention is produced as a fusion protein with a Flag peptide, and affinity chromatography using an anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], and can also be purified by affinity chromatography using a metal coordination resin produced as a fusion protein with polyhistidine and having high affinity with polyhistidine. Furthermore, it can also be purified by affinity chromatography using an antibody against the protein itself.
- the protein of the present invention is produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid sequence information of the protein obtained above. be able to. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.
- chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.
- the protein of the present invention has the property of producing hydrogen peroxide by acting on a glycated peptide produced from a glycated protein by the action of a proteolytic enzyme on the glycated protein. It can be used for the measurement of glycated protein in various samples. Specifically, a substance is produced by reacting a sample with a proteolytic enzyme to produce a glycated peptide, reacting the produced glycated peptide with the protein of the present invention, and reacting the glycated peptide with the protein of the present invention.
- the glycated protein in the sample can be measured by measuring a substance consumed in the reaction between the glycated peptide and the protein of the present invention.
- the reaction relating to the measurement of glycated protein in a sample may be performed in an aqueous medium described later.
- the glycated protein in the present invention include glycated hemoglobin such as hemoglobin A1c, glycated albumin, etc., glycated hemoglobin is preferred, and hemoglobin A1c is particularly preferred.
- the measuring method of the present invention will be described.
- the sample used in the measurement method of the present invention is not particularly limited as long as it contains a glycated protein.
- whole blood, plasma, serum, blood cells, cell samples, urine, spinal fluid, sweat, tears, saliva examples include biological samples such as skin, mucous membrane, and hair, and foods.
- whole blood, plasma, serum, blood cells and the like are preferable, and whole blood, blood cells and the like are particularly preferable.
- the whole blood includes a sample in which plasma is mixed with a blood cell fraction derived from whole blood. These samples may be subjected to pretreatment such as hemolysis, separation, dilution, concentration and purification.
- Hemoglobin is a tetramer with a molecular weight of 64,500 consisting of two polypeptides of ⁇ chain and ⁇ chain.
- the N-terminal 3 amino acid sequence of the ⁇ chain of hemoglobin is valine-leucine-serine, and the N-terminal 3 amino acid sequence of the ⁇ chain is valine-histidine-leucine.
- Hemoglobin A1c is defined as a glycated N-terminal valine of the ⁇ chain. Hemoglobin is known to have multiple glycation sites in the molecule (The Journal of Biological Chemistry (1980), 256, 3120-3127).
- ⁇ -fructosylvaline (hereinafter abbreviated as ⁇ -FV) and ⁇ derived from glycated hemoglobin in which the ⁇ -chain N-terminal valine residue is glycated by allowing a protease to act on a sample containing glycated hemoglobin and ⁇ -FVH, ⁇ -FV and ⁇ -fructosylvalyl leucine (hereinafter abbreviated as ⁇ -FVL), ⁇ chain and / or ⁇ chain derived from glycated hemoglobin in which the ⁇ -chain N-terminal valine residue is glycated A glycated amino acid and / or glycated oligopeptide such as ⁇ -FK derived from glycation of the ⁇ -amino group of the internal lysine residue is produced.
- ⁇ -FV ⁇ -fructosylvaline
- ⁇ -FV
- a glycated amino acid such as ⁇ -FK is also produced from a glycated protein other than glycated hemoglobin in the whole blood such as glycated albumin. That is, when a proteolytic enzyme is allowed to act on a sample containing purified hemoglobin or a sample containing whole blood, for example, ⁇ -FVH, ⁇ -FV, ⁇ -FK, ⁇ -FVL, etc. are generated, and ⁇ -FVH and ⁇ -FVL are produced. Is derived from glycated hemoglobin, and ⁇ -FVH is specifically derived from hemoglobin A1c.
- ⁇ -FVH when measuring hemoglobin A1c, ⁇ -FVH may be specifically measured. Since the protein of the present invention has high reactivity with ⁇ -FVH and low reactivity with ⁇ -FK, hemoglobin A1c can be measured effectively.
- proteolytic enzyme Any proteolytic enzyme that can be used in the present invention may be used as long as it acts on the glycated protein to be measured contained in the sample.
- a protease derived from an animal, a plant or a microorganism, a metalloprotease, endo Examples include protease, exoprotease, serine protease, cysteine protease, acidic protease, alkaline protease, thiol protease and the like.
- animal-derived proteases include elastase, trypsin, chymotrypsin, pepsin, bovine pancreatic protease, porcine liver leucine aminopeptidase, cathepsin, calpain, protease Type-I, protease type-XX (above, manufactured by Sigma), aminopeptidase M (Aminopeptidase M), carboxypeptidase A (Carboxypeptidase A) (above, made by Boehringer Mannheim), pancreatin (Pancreatin: Wako Pure Chemical Industries, Ltd.) Manufactured by Sigma).
- plant-derived proteases include kallikrein, ficin, papain, chymopapain, bromelain, carboxypeptidase W (above, Sigma), papain W-40, bromelain F And Amano Enzyme).
- microorganism-derived protease examples include the following (1) to (14).
- Penicillium-derived protease PD enzyme (manufactured by Kikkoman), protease B “Amano” (manufactured by Amano Enzyme), deoxyn 1 (manufactured by Nagase ChemteX), and the like.
- protease type XIV also known as Pronase
- protease-XXI above, Sigma
- actinase-AS above, actinase-AF
- actinase-E above, Kaken Pharmaceutical
- Alkalofilic Proteinase Toyobo
- Pronase E Roche, Calbiochem-Nobiochem, Sigma
- Pronase Boehringer Mannheim
- protease type XVII manufactured by Sigma
- endoproteinase Glu-C manufactured by Boehringer Mannheim
- V8 protease manufactured by Takara, manufactured by Wako Pure Chemical Industries, Ltd.
- Protease derived from yeast Yeast
- proteinase A Proteinase A, manufactured by Sigma
- carboxypeptidase Y Carboxypeptidase Y, manufactured by Boehringer Mannheim
- proteases derived from the genera Bacillus, Aspergillus, Streptomyces, and Trityratium are preferred because they have a large effect on human hemoglobin, and proteases derived from the genus Bacillus are particularly preferred.
- metalloproteases include thermolysin and protease N.
- the endoprotease include thermolysin, papain, subtilisin, pepsin, trypsin, chymotrypsin and the like.
- the exoprotease include aminopeptidase and carboxypeptidase.
- serine proteases include thermitase, proteinase K, trypsin, chymotrypsin, thrombin, plasmin, and elastase.
- cysteine proteases include papain and caspase.
- acidic proteases include pepsin and cathepsin D.
- alkaline protease include orientase 22BF.
- thiol protease include papain, ficin, bromelain and the like.
- the concentration of the proteolytic enzyme is preferably 0.01 to 100,000 ⁇ U / mL, more preferably 0.1 to 10,000 ⁇ U / mL in the reaction solution.
- two or more kinds of enzymes can be used in combination.
- the proteolytic enzyme used in the present invention is preferably uncolored.
- an absorbance at a wavelength of 300 to 800 nm is preferably 100 ⁇ mAb or less. More preferred is mAbs. It is preferable to use a proteolytic enzyme that has been purified by various chromatograms, salting out, dialysis, activated carbon treatment or the like to reduce the above-mentioned absorbance.
- the concentration of the enzyme which is the protein of the present invention is preferably 0.01 to 1,000 ⁇ U / mL, more preferably 0.1 to 100 ⁇ U / mL in the reaction solution.
- the glycated protein to be measured in the sample of the present invention can be measured by sequentially performing the following steps (i) to (iii). (i) reacting a sample with a proteolytic enzyme to produce a glycated peptide; (ii) reacting the produced glycated peptide with the protein of the present invention, and (iii) A step of measuring the substance produced or consumed in the step (ii).
- the above steps (i) to (iii) may be performed in an aqueous medium.
- the aqueous medium include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable.
- the buffer used in the buffer include tris (hydroxymethyl) aminomethane buffer (Tris buffer), phosphate buffer, borate buffer, Good's buffer, and the like.
- Good buffering agents include, for example, 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA) Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) ), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfone Acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane Sulfonic acid (HEPE
- the concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L.
- the reaction temperature of the reaction in each step is, for example, 10 to 50 ° C., preferably 20 to 40 ° C., and the reaction time is 1 second to 60 minutes, preferably 1 to 10 minutes.
- step (ii) If the proteolytic enzyme does not affect the reaction of step (ii), it does not need to be deactivated after step (i), but heating, cooling, centrifugation, membrane filtration, addition of inhibitors
- the enzyme can also be prevented from acting in step (ii) by, for example.
- the products generated in the reaction solution by the reaction between the glycated peptide and the protein having fructosyl peptide oxidase activity of the present invention include hydrogen peroxide, sugar osone ( ⁇ -ketoaldehyde), peptide Etc.
- examples of the substance consumed by the reaction between the glycated peptide and the protein having fructosyl peptide oxidase activity of the present invention include oxygen molecules.
- the oxygen molecules consumed in the step (ii) are measured by, for example, an electrochemical measurement method using an oxygen electrode.
- the hydrogen peroxide produced in step (ii) of the present invention can be measured using, for example, an optical technique or an electrochemical technique.
- the optical method include an absorbance method and a luminescence method.
- Specific examples include an optical measurement method using a hydrogen peroxide measurement reagent, an electrochemical measurement method using a hydrogen peroxide electrode, and the like.
- the hydrogen peroxide measuring reagent is a reagent for converting the generated hydrogen peroxide into a detectable substance.
- the detectable substance include a dye and light, but a dye is preferable.
- the hydrogen peroxide measurement reagent contains an oxidation coloring substance chromogen and a peroxidation active substance such as peroxidase.
- the oxidative coloring chromogen include an oxidative coupling chromogen described later and a leuco chromogen described later.
- the hydrogen peroxide measuring reagent contains a chemiluminescent substance.
- the chemiluminescent substance includes a bioluminescent substance, and examples thereof include luminol, isoluminol, lucigenin, acridinium ester, and oxalate ester.
- hydrogen peroxide When using a reagent containing an oxidative coloring chromogen and a peroxidase active substance such as peroxidase as a hydrogen peroxide measuring reagent, hydrogen peroxide is oxidized in the presence of the peroxidative active substance. It is possible to measure hydrogen peroxide by reacting with, producing a dye and measuring the produced dye.
- a reagent for measuring hydrogen peroxide containing a chemiluminescent substance When a reagent for measuring hydrogen peroxide containing a chemiluminescent substance is used, hydrogen peroxide can be measured by reacting hydrogen peroxide with the chemiluminescent substance to generate photons and measuring the generated photons. it can.
- An oxidative coupling type chromogen is a chromogen that reacts with hydrogen peroxide in the presence of a peroxidase active substance such as peroxidase to produce a dye by an oxidative coupling reaction.
- a peroxidase active substance such as peroxidase
- Specific examples of the oxidative coupling type chromogen include couplers such as 4-aminoantipyrine, phenolic or aniline hydrogen donors, and the like.
- a coupler and a phenol-based or aniline-based hydrogen donor compound are oxidatively coupled in the presence of hydrogen peroxide and a peroxide active substance to form a dye.
- couplers examples include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone.
- phenolic hydrogen donor examples include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.
- N- (3-sulfopropyl) aniline N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl -N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methyl Aniline, N, N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-meth
- the leuco chromogen is a chromogen that reacts with hydrogen peroxide in the presence of a peroxidase active substance such as peroxidase to produce a pigment alone.
- a peroxidase active substance such as peroxidase to produce a pigment alone.
- CCAP 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine
- MCDP 10-N-methylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine
- DA-64 N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt
- DA-67 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) Phenothiazine sodium salt
- DA-67 4,4′-bis (dimethylamin
- the concentration of the peroxidation active substance is not particularly limited as long as it is suitable for the measurement. More preferred is -50 U / mL.
- the concentration of the oxidative coloring type chromogen is not particularly limited as long as it is suitable for measurement, but is preferably 0.01 to 10 g / L, and more preferably 0.02 to 5 g / L.
- the electrode to be used is not particularly limited as long as it is a material that exchanges electrons with hydrogen peroxide. Examples thereof include platinum, gold, and silver. .
- known methods such as amperometry, potentiometry, coulometry, and the like can be used.
- An electron carrier can be interposed in the reaction between the oxidase or substrate and the electrode, and the resulting oxidation, reduction current, or electric quantity thereof can be measured.
- any substance having an electron transfer function can be used, and examples thereof include substances such as ferrocene derivatives and quinone derivatives.
- the oxidation, reduction current, or electric quantity thereof obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode can be measured.
- step (ii) sugar oxone ( ⁇ -ketoaldehyde form) is produced together with hydrogen peroxide. Therefore, hemoglobin A1c in the sample is also measured by measuring the produced sugar oxone ( ⁇ -ketoaldehyde form). can do. By measuring hydrogen peroxide produced by the action of glucose oxidase on the ⁇ -ketoaldehyde body, it can be measured with high sensitivity (Japanese Patent Laid-Open No. 2000-333696).
- sample preparation method The sample containing the glycated protein to be measured can be separated from the biological sample as necessary.
- the separation method include centrifugation, filtration, and a method using a blood cell separation membrane.
- the separation method by centrifugation can separate whole blood into blood cells, plasma or serum.
- the blood cells can be washed with an isotonic solution such as physiological saline to obtain washed blood cells from which plasma-derived components have been removed.
- a sample containing blood cells such as whole blood, blood cells, and washed blood cells can be diluted with a hypotonic solution for hemolysis.
- the hypotonic solution may be any solution as long as it can lyse blood cells, but includes water, buffer solution, and the like, and preferably contains an additive such as a surfactant.
- the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants.
- Examples of methods for preparing washed blood cells include the following methods. Blood is collected from healthy individuals and diabetics, mixed by inversion, and then centrifuged (3,000 rpm) at 25 ° C for 5 minutes. After centrifugation, the supernatant plasma is removed. Add 4 volumes of physiological saline to 1 volume of blood cell layer in the lower layer, mix by inversion, and centrifuge (3,000 rpm) at 25 ° C for 5 minutes. After centrifugation, the supernatant saline is removed. After repeating this washing operation three times, 9 volumes of distilled water can be added to 1 volume of the washed blood cell layer to obtain washed blood cells.
- the reagent for measuring glycated protein and the measuring kit of the present invention can be used in the method for measuring glycated protein of the present invention.
- the reagent for measuring glycated protein of the present invention can take the form of a kit as a form suitable for storage, transportation and distribution. Examples of the kit include a two-reagent system, a three-reagent system, and the like.
- the reagent for measuring a glycated protein of the present invention includes a proteolytic enzyme and a protein having fructosyl peptide oxidase activity of the present invention. Furthermore, the reagent for measuring a glycated protein of the present invention may include a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from the glycated protein. Examples of the product produced by the reaction between the protein of the present invention and a glycated peptide produced from a glycated protein include hydrogen peroxide, sugar osone ( ⁇ -ketoaldehyde), peptide and the like.
- Examples of the reagent for measuring a product produced by the reaction of the protein of the present invention with a glycated peptide produced from a glycated protein include, for example, a reagent for measuring hydrogen peroxide, a reagent for measuring sugar osone ( ⁇ -ketoaldehyde), Peptide (Val-His) measurement reagents and the like can be mentioned, and hydrogen peroxide measurement reagents are preferred.
- kits for measuring a glycated protein to be measured of the present invention include the kits of the following embodiments.
- Kit 1 (2-reagent kit) A kit containing the following two reagents. (1) a reagent containing a proteolytic enzyme; (2) A reagent containing the protein of the present invention.
- Kit 2 (2-reagent kit) A kit containing the following two reagents. (1) a reagent containing a proteolytic enzyme; (2) A reagent comprising a reagent for measuring the protein of the present invention and a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- Kit 3 A kit containing the following two reagents. (1) a reagent comprising a proteolytic enzyme and a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein; (2) A reagent containing the protein of the present invention.
- Kit 4 A kit containing the following two reagents. (1) a reagent comprising a proteolytic enzyme and a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein; (2) A reagent comprising a reagent for measuring the protein of the present invention and a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- Kit 5 (3-reagent kit) A kit containing the following three reagents. (1) a reagent containing a proteolytic enzyme; (2) a reagent containing the protein of the present invention; and (3) A reagent comprising a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- Kit 6 (3-reagent kit) A kit containing the following three reagents. (1) a reagent comprising a proteolytic enzyme and a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein; (2) a reagent containing the protein of the present invention; and (3) A reagent comprising a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- Kit 7 (3-reagent kit) A kit containing the following three reagents. (1) a reagent containing a proteolytic enzyme; (2) a reagent comprising the protein of the present invention and a reagent for measuring a product produced by the reaction of the protein of the present invention and a glycated peptide produced from the glycated protein; and (3) A reagent comprising a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- Kit 8 (3-reagent kit) A kit containing the following three reagents. (1) a reagent comprising a proteolytic enzyme and a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein; (2) a reagent comprising the protein of the present invention and a reagent for measuring a product produced by the reaction of the protein of the present invention and a glycated peptide produced from the glycated protein; and (3) A reagent comprising a reagent for measuring a product produced by a reaction between the protein of the present invention and a glycated peptide produced from a glycated protein.
- the protein of the present invention the glycated protein, and the product produced by the reaction of the protein of the present invention and the glycated peptide produced from the glycated protein
- the reagents include those described above.
- the reagent for measuring the product produced by the reaction between the protein of the present invention and the glycated peptide produced from the glycated protein is a hydrogen peroxide measuring reagent
- the hydrogen peroxide measuring reagent include the above-mentioned excess. Examples thereof include a reagent for measuring hydrogen oxide.
- the coupler and the phenolic or aniline hydrogen donor may be contained in the same reagent, but are contained in separate reagents. It is preferred that
- the measurement reagent and measurement kit of the present invention may further contain a measurement standard substance such as a standard protein.
- the measurement reagent and measurement kit of the present invention may each contain a buffer, a stabilizer, a preservative, an influence substance removing agent, a nonspecific reaction inhibitor, a surfactant, and the like, if necessary.
- the buffering agent include the aforementioned buffering agents.
- the stabilizer include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride, amino acids, salts such as albumin, dextran, and calcium acetate.
- Examples of the preservative include sodium azide and antibiotics.
- Examples of the influence substance removing agent include ascorbate oxidase for eliminating the influence of ascorbic acid.
- the nonspecific reaction inhibitor include polymer compounds such as dextran sulfate.
- the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants.
- the measurement reagent and measurement kit of the present invention may be lyophilized or dissolved in a reaction solution.
- the kit can be used by dissolving in the aforementioned aqueous medium or reaction solution.
- a reagent for dissolving a lyophilized reagent may be contained in the kit as necessary.
- the content of the proteolytic enzyme in the measurement kit of the present invention is preferably such that the concentration when dissolved in an aqueous medium is 0.01 to 1,000,000 U / mL, more preferably 0.1 to 100,000 U / mL. .
- the content of the protein of the present invention in the measurement kit of the present invention is preferably such that the concentration in a state dissolved in an aqueous medium is 0.01 to 10,000 U / mL, more preferably 0.1 to 1,000 U / mL. .
- the content of peroxidase and the oxidative coupling type chromogen was dissolved in an aqueous medium, respectively.
- the content is preferably 1 to 600 U / mL and 0.5 to 40 g / L, more preferably 2 to 150 U / mL and 1 to 20 g / L.
- Example 1 Construction of fructosyl peptide oxidase gene expression system
- the Emericella nidulans KY125 strain was selected as a bacterium that produces an enzyme with relatively high activity against ⁇ -FVH and low activity against ⁇ -FK.
- a primer SEQ ID NO: 5, SEQ ID NO: 5, which is a strain of an Aspergillus nidulans FGSC A4 strain which is an E. nidulans- related bacterium and whose entire genome sequence has been decoded.
- SEQ ID NO: 6 the full length of the fructosyl peptide oxidase gene was amplified using the PCR method.
- the fructosyl peptide oxidase gene of the KY125 strain contained 5 introns among 6 exons. Therefore, only the exon portion was amplified by overlapping PCR method (Nucleic Acids Res., 16, 7351 (1988)), and they were combined to construct a mature fructosyl peptide oxidase gene consisting only of exons. As a result of decoding the DNA sequence, it was found that the mature fructosyl peptide oxidase gene consists of 1317 bp and 438 amino acids.
- fructosyl peptide oxidase gene of E. nidulans KY125 strain the fructosyl peptide oxidase obtained above was placed on the restriction enzyme sites of NcoI and BamHI of the expression vector pTrc99A (4,176-bp, GE Japan). The gene was inserted.
- the recombinant DNA (plasmid) (pTrcFPOX-1) was used to transform E. coli XL1-Blue (Funakoshi). By culturing this transformant overnight in an LB medium containing 50 mg / L of ampicillin, a protein having fructosyl peptide oxidase activity was obtained in the cells.
- Example 2 Construction of fructosyl peptide oxidase A random mutation was introduced into the fructosyl peptide oxidase gene obtained in Example 1. Random mutations were introduced using Stratagene's “GeneMorph II Random Mutagenesis Kit”. By using PCR with pTrcFPOX-1 as template DNA and using primers (SEQ ID NO: 5 and SEQ ID NO: 6) corresponding to the regions corresponding to the 5′-upstream and 3′-downstream of the fructosyl peptide oxidase gene, Substitution was introduced and full length amplification was performed simultaneously.
- the PCR product was cleaved with NcoI and BamHI, purified with PureLink PCR Purification Kit (Invitrogen), ligated to the NcoI and BamHI sites of pTrc99A, and then transformed into E.coli XL1-Blue strain.
- a colony (transformant) that had grown by overnight culture on a plate containing LB medium containing 50 mg / L of ampicillin was picked up. They were cultured at 30 ° C. for 18 hours using 24-well culture plates (Sumitomo Bakelite) containing 2 ⁇ mL of LB medium containing 50 ⁇ mg / L of ampicillin. BugBuster (manufactured by Novagen) was added to the culture solution, and after lysis and centrifugation, the supernatant was used as an enzyme source to measure enzyme activity against two types of substrates ( ⁇ -FK, ⁇ -FVH).
- a plasmid was prepared from each transformant, and the base sequence of the fructosyl peptide oxidase gene portion was decoded using a DNA sequencer. Then, changes in enzyme activity, heat resistance and substrate specificity were correlated with changes in the base sequence (amino acid sequence).
- fructosyl peptide oxidase with increased ⁇ -FVH activity or heat resistance in the fructosyl peptide oxidase obtained in Example 1, Ser at position 71 was replaced with Tyr, and Lys at position 109 was replaced with Arg.
- the fructosyl peptide oxidase (hereinafter referred to as FPOX-9) was obtained by replacing the 94th Ile with Met, the 269th Phe with Ile, and the 104th Glu with Lys.
- the amino acid sequence of FPOX-9 is shown in SEQ ID NO: 1, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 2.
- FPOX-9 FPOX-10 in which the 59th Ser was replaced with Gly, 58th Met of the FPOX-10 was replaced with Phe, and FPOX-11 in which the 105th Gly was replaced with Lys, FPOX-13 in which 183th Gly of FPOX-11 was replaced with Glu and FPOX-14 in which 302th Pro of FPOX-13 was replaced with Leu were obtained.
- FPOX-15 was obtained by substituting Asp at the 272nd Asn of the FPOX-14.
- the amino acid sequence of FPOX-15 is shown in SEQ ID NO: 3, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 4, respectively.
- Table 1 above shows the substrate selectivity and heat resistance of various mutant fructosyl peptide oxidases.
- ⁇ -FVH activity and ⁇ -FK activity represent relative values when the ⁇ -FK activity of FPOX-9 is 1.00, and the number of additional mutations represents the number of substitutions from FPOX-9. To express.
- Example 3 Acquisition of mutant fructosyl peptide oxidase Test tube containing 10 mL of LB medium containing 50 mg / L of ampicillin from E. coli XL1-Blue strain containing FPOX-9 obtained in Example 2 Ten cells were inoculated and cultured with shaking at 30 ° C. for 24 hours. These culture solutions were respectively transferred to 10 Erlenmeyer flasks containing 300 mL of LB medium containing 50 mg / L of ampicillin and 20 mg / L of IPTG, and cultured with shaking at 30 ° C. for 24 hours.
- Approximately 3,000 ⁇ mL of the culture solution was collected, and the cells were collected by centrifugation at 10,000 ⁇ x ⁇ g for 15 minutes.
- the cells were suspended in about 50 ⁇ mL of 10 ⁇ mmol / L phosphoric acid buffer (pH 7.0), and disrupted using an ultrasonic disrupter for 1 minute under ice cooling. Crushing under the same conditions was further repeated 9 times.
- the cell disruption solution was centrifuged at 10,000 ⁇ xg for 15 minutes, and the resulting supernatant was used as a crude enzyme extract.
- Solid ammonium sulfate was added to the crude enzyme extract so as to be 60% saturated, and the mixture was allowed to stand under ice cooling for 2 hours to sufficiently precipitate the target enzyme protein. Then, the precipitate was recovered by centrifugation at 10,000 x g for 15 minutes. The precipitate was dissolved in about 20 mL of 10 mL / L phosphate buffer (pH 7.0), and the solution was dialyzed against 5,000 mL of the same buffer overnight in a cold place.
- the dialyzed enzyme solution is packed with DEAE-Toyopearl (Toyobo Co., Ltd.) in a 10 ⁇ x 100 cm column, passed through a column pre-equilibrated with 10 mmol / L phosphate buffer (pH 7.0), and the same buffer. Further washing with liquid.
- the target enzyme passed through without being adsorbed on the column. On the other hand, most of the contaminating proteins were adsorbed on the column. As a result, as shown in Table 2, the target enzyme was purified about 50 times with a yield of 31%.
- E. coli XL1-Blue strain having FPOX-15 obtained in Example 2 was subjected to a series of purification steps to obtain a solution of purified FPOX-15.
- Table 2 above shows the state of FPOX-9 in each step of purification of fructosyl peptide oxidase FPOX-9.
- Second reagent Tris buffer (pH 7.5) 100 mmol / L Fructosyl peptide oxidase FPOX-9 2 ⁇ L 4-Aminoantipyrine 0.5 mmol / L TOOS 0.2 mmol / L Peroxidase (from horseradish) 10 U / mL
- the FPOX-9 solution prepared in Example 3 was used.
- glycated peptide ⁇ -FVH and glycated amino acids ⁇ -FK and ⁇ -FV were used as substrates.
- the ⁇ -FVH solution and the ⁇ -FV solution are prepared using a Tris buffer so that each concentration is 0, 0.05, 0.1, 0.5, 1, 2, 5, 10 mmol / L in the third reagent. did.
- the ⁇ -FK solution was prepared using a Tris buffer so as to have respective concentrations of 0, 1, 2, 5, 10, 20, 40, 50, 80, and 100 mmol / L in the third reagent.
- the substrate concentration (mmol / L) is plotted on the horizontal axis, and the enzyme activity (U / mL) corresponding to the vertical axis is plotted.
- the substrate concentration corresponding to / 2 was calculated as the Km value (Michaelis constant). The results are shown in Table 3.
- Table 3 above shows Km values for various substrates of fructosyl peptide oxidase FPOX-9.
- the fructosyl peptide oxidase of the present invention was shown to be an enzyme that has high substrate specificity for ⁇ -FVH and ⁇ -FV and is difficult to react with ⁇ -FK.
- the PCR product was cleaved with NcoI and BamHI, purified with PureLink PCR Purification Kit (Invitrogen), ligated to the NcoI and BamHI sites of pTrc99A, and then transformed into E.coli XL1-Blue strain.
- a colony (transformant) that had grown by overnight culture on a plate containing LB medium containing 50 mg / L of ampicillin was picked up. They were cultured at 30 ° C. for 18 hours using 24-well culture plates (Sumitomo Bakelite) containing 2 ⁇ mL of LB medium containing 50 ⁇ mg / L of ampicillin. BugBuster (manufactured by Novagen) was added to the culture solution, and after lysis and centrifugation, the supernatant was used as an enzyme source to measure enzyme activity against two types of substrates ( ⁇ -FK, ⁇ -FVH).
- ⁇ -FVH activity and ⁇ -FK activity represent relative values when the ⁇ -FK activity of FPOX-9 is 1.00. From Table 4, it can be seen that the ratio of ⁇ -FVH activity to ⁇ -FK activity (FVH / FK) does not change compared to FPOX-9 even when the amino acid of FPOX-9 is mutated. Or it became clear that it improved.
- FPOX-15 First reagent sodium dihydrogen phosphate (pH 8.0) 100 mmol / L Fructosyl peptide oxidase FPOX-15 2 ⁇ L
- FPOX-15 solution a solution obtained by diluting the solution obtained in Example 3 four times was used.
- Second reagent sodium dihydrogen phosphate (pH 6.0 or pH 7.0 or pH 8.0) 100 mmol / L Peroxidase (from horseradish) 3U / mL 4-Aminoantipyrine 0.5mmol / L EMSE 0.4mmol / L
- glycated peptide ⁇ -FVH and glycated amino acids ⁇ -FK and ⁇ -FV were used as substrates.
- ⁇ -FVH solution and ⁇ -FV solution were prepared using purified water so as to have respective concentrations of 0, 2, 2.5, 3, 3.66 mmol / L in the third reagent.
- the ⁇ -FK solution was prepared using purified water so as to have concentrations of 0, 20, 30, 40, and 50 mmol / L in the third reagent.
- the horizontal axis represents the substrate concentration (mmol / L) and the vertical axis represents the corresponding enzyme activity (U / mL).
- the substrate concentration corresponding to / 2 was calculated as the Km value (Michaelis constant). The results are shown in Table 5.
- Table 5 above shows Km values for various substrates of fructosyl peptide oxidase FPOX-15.
- the fructosyl peptide oxidase of the present invention was shown to be an enzyme having high substrate specificity for ⁇ -FVH and ⁇ -FV and low substrate specificity for ⁇ -FK.
- Example 7 Isoelectric point pI of fructosyl peptide oxidase (FPOX-9 and FPOX-15) Each solution (3 ⁇ L) of 1 mg / mL phosphate buffer (pH 7.0) solution of FPOX-9 and FPOX-15 was subjected to isoelectric focusing gel of Phast System (fully automated electrophoresis system, GE Healthcare). In accordance with the operation procedure, electrophoresis, staining, and decolorization operation were performed to determine the pI of each fructosyl peptide oxidase. The results are shown in Table 6.
- Example 8 Effect of pH on the stability of fructosyl peptide oxidases FPOX-9 and FPOX-15
- the effect of pH on the stability of fructosyl peptide oxidases FPOX-9 and FPOX-15 was determined as follows. Evaluation was made using three reagents. In the evaluation, a reagent immediately after preparation, a reagent stored at 30 ° C. for 24 hours (one day), and a reagent stored at 30 ° C. for 5 days were used as the first reagent.
- Second reagent sodium dihydrogen phosphate (pH 6.0 or pH 7.0 or pH 8.0) 100 mmol / L Peroxidase (from horseradish) 3 U / mL 4-Aminoantipyrine 0.5 mmol / L EMSE 0.4 mmol / L
- glycated amino acid ⁇ -FG was used as a substrate.
- Table 7 shows the effect of pH on the stability of fructosyl peptide oxidase FPOX-9
- Table 8 shows the effect of pH on the stability of fructosyl peptide oxidase FPOX-15.
- the fructosyl peptide oxidase FAOX-9 and FPOX-15 of the present invention are stable under any pH of pH 5.0, pH 6.0, pH 7.0 and pH 8.0. It turned out to be.
- Example 9 Effect of metal on stability of fructosyl peptide oxidase FPOX-9 and FPOX-15
- the effect of metal on the stability of fructosyl peptide oxidase FPOX-9 and FPOX-15 was measured using the following first reagent to second reagent. Evaluation was made using three reagents. In the evaluation, a reagent immediately after preparation, a reagent stored at 5 ° C. for 24 hours (one day), and a reagent stored at 30 ° C. for 24 hours (one day) were used as the first reagent.
- the FPOX-9 solution used was a solution obtained by diluting the solution obtained in Example 3 20 times
- the FPOX-15 solution used was a solution obtained by diluting the solution obtained in Example 3 100 times.
- glycated amino acid ⁇ -FG was used as a substrate.
- Table 9 shows the influence of metal on the stability of fructosyl peptide oxidase FPOX-9
- Table 10 shows the influence of metal on the stability of fructosyl peptide oxidase FPOX-15.
- a novel protein useful for diagnosis of lifestyle-related diseases such as diabetes, DNA encoding the protein, a method for producing the protein, a method for measuring a glycated protein using the protein, and the protein A reagent for measuring glycated protein is provided.
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Abstract
Description
フルクトシルペプチドオキシダーゼは、細菌、真菌、植物から見出されている。例えば、アカエトミエラ属、カエトミウム属(特許文献3)、カーブラリア属(特許文献2)、バラ科、ブドウ科、セリ科(特許文献4)、ショウガ科(特許文献5)等由来のフルクトシルペプチドオキシダーゼが知られている。
(1)α-糖化アミノ酸(例えば、α-フルクトシルバリン)に比べて、α-糖化ジペプチド(α-フルクトシルバリルヒスチジン)に対する活性は、必ずしも高くないこと、
(2)上述のように、N末端のα-糖化ジペプチド以外にも、リジンのε-アミノ基に糖が結合したε-糖化アミノ酸(ε-フルクトシルリジン)にも作用し、ヘモグロビンA1c測定における実測値を増加させること、
(3)酵素を用いる測定法の場合、測定時や保存時において酵素が不安定となる、
等の欠点があった。
(1) 以下の[1]~[4]のいずれかに記載の蛋白質。
[1]配列番号1で表されるアミノ酸配列を有する蛋白質
[2]配列番号1で表されるアミノ酸配列において、1以上のアミノ酸が欠失、置換または付加したアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質
[3]配列番号1で表されるアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質
[4]寄託番号FERM BP-11026として寄託された大腸菌XL1-Blue MRF'株が担持する発現プラスミドによってコードされるフルクトシルペプチドオキシダーゼ活性を有する蛋白質
(2) 配列番号3で表されるアミノ酸配列からなる、(1)に記載の蛋白質。
(3) 以下の[1]~[3]のいずれかに記載のDNA。
[1](1)記載の蛋白質をコードするDNA
[2]配列番号2で表される塩基配列を有するDNA
[3]配列番号2で表される塩基配列と相補的な塩基配列を有するDNAとストリンジェントな条件下でハイブリダイズし、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質をコードするDNA
(4) 配列番号3で表されるアミノ酸配列からなる蛋白質をコードする、(3)に記載のDNA。
(5) 配列番号4で表される塩基配列からなる、(3)に記載のDNA。
(6) (3)~(5)のいずれかに記載のDNAを含有する組換え体DNA。
(7) (6)記載の組換え体DNAを有する形質転換体。
(8) (7)記載の形質転換体を培地に培養し、培養物中に該蛋白質を生成、蓄積させ、該培養物より該蛋白質を採取する(1)又は(2)記載の蛋白質の製造方法。
(9) 試料を蛋白質分解酵素と反応させて糖化ペプチドを生成させ、次いで、生成した糖化ペプチドを(1)又は(2)記載の蛋白質と反応させ、当該糖化ペプチドと当該蛋白質との反応により生成した物質又は当該糖化ペプチドと当該蛋白質との反応において消費された物質を測定することを特徴とする、試料中の糖化蛋白質の測定方法。
(10) 糖化蛋白質が、糖化ヘモグロビンである(9)記載の測定方法。
(11) 糖化ヘモグロビンが、ヘモグロビンA1cである(10)記載の測定方法。
(12) 蛋白質分解酵素、(1)又は(2)記載の蛋白質を含む糖化蛋白質測定用試薬。
(13) さらに、(1)又は(2)記載の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む(12)記載の試薬。
(14) 生成物が、過酸化水素である(13)記載の試薬。
(15) 糖化蛋白質が、糖化ヘモグロビンである(12)~(14)のいずれかに記載の試薬。
(16) 糖化ヘモグロビンが、ヘモグロビンA1cである(15)記載の試薬。
(17) 寄託番号FERM BP-11026として寄託された大腸菌XL1-Blue MRF'株。
本発明の蛋白質としては、
[1]配列番号1で表されるアミノ酸配列を有する蛋白質、
[2]配列番号1で表されるアミノ酸配列において、1以上のアミノ酸が欠失、置換または付加したアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質、
[3]配列番号1で表されるアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質、および、
[4]寄託番号FERM BP-11026として寄託された大腸菌XL1-Blue MRF'株が担持する発現プラスミドによってコードされるフルクトシルペプチドオキシダーゼ活性を有する蛋白質
を挙げることができる。
A群:ロイシン、イソロイシン、ノルロイシン、バリン、ノルバリン、アラニン、2-アミノブタン酸、メチオニン、O-メチルセリン、t-ブチルグリシン、t-ブチルアラニン、シクロヘキシルアラニン
B群:アスパラギン酸、グルタミン酸、イソアスパラギン酸、イソグルタミン酸、2-アミノアジピン酸、2-アミノスベリン酸
C群:アスパラギン、グルタミン
D群:リジン、アルギニン、オルニチン、2,4-ジアミノブタン酸、2,3-ジアミノプロピオン酸
E群:プロリン、3-ヒドロキシプロリン、4-ヒドロキシプロリン
F群:セリン、スレオニン、ホモセリン
G群:フェニルアラニン、チロシン
(a) 作用:分子状酸素を用いて、糖化ペプチドを酸化して、糖オソン(α-ケトアルデヒド体)、ペプチド及び過酸化水素を生成する。
(b) 基質特異性:α-FVHに対する反応性が高く、かつε-フルクトシルリジン(以下、ε-FKと略記する)に対する反応性が低い。
作用適温の範囲については、特に制限はないが、30~50℃付近が好ましい。耐熱性は、高い方が好ましく、例えば、50℃、15分間の熱処理後の残存活性が、25%以上のものが好ましく用いられる。
A液:発色液
A-1液:4-アミノアンチピリンを2.4 mmol/L濃度になるように、イオン交換水に溶解する。
A-2液:N-エチル-N-(3-メチルフェニル)-N’-サクシニルエチレンジアミン(EMSE)を32 mmol/L濃度になるように、イオン交換水に溶解する。
B液:パーオキシダーゼ溶液
パーオキシダーゼ(110 U/mg、東洋紡績社製)を2 mg/mL濃度になるように、0.1 mol/Lリン酸緩衝液(pH 7.0)に溶解する。
C液:基質溶液
α-FVHまたはε-FK(ペプチド研究所製)を10 mmol/L濃度になるように、0.1 mol/Lリン酸緩衝液(pH 7.0)に溶解する。
A-1液 50μL、A-2液 50μL、B液 2μL、C液 20μLを混合し、水で200μLにフィルアップした後、30℃にて5分間プレインキュベーションした後、酵素液 1μLを添加し、30℃で30分間反応させ、プレートリーダー(infinite F200、Tecan社製)で550 nmにおける吸光度を測定した。なお、ブランク値は、基質溶液(C液)の代わりにイオン交換水を用いたものを測定する。
そして前記の測定系に、種々の量の過酸化水素を添加して、550 nmの吸光度を測定し、過酸水素の量と吸光度の関係を表わす検量曲線を作成し、そこから酵素の単位数(酵素力価)を求める。
(a)寄託機関の名称・あて名
名称:独立行政法人産業技術総合研究所 特許生物寄託センター
あて名:日本国茨城県つくば市東1丁目1番地1 中央第6(郵便番号305-8566)
(b)受領日(寄託日):2008年9月19日
(c)受領番号(寄託番号): FERM BP-11026
本発明のDNAとしては、
[1]上記1の[1]~[3]の本発明の蛋白質をコードするDNA、
[2]配列番号2で表される塩基配列を有するDNA、および
[3]配列番号2で表される塩基配列と相補的な塩基配列を有するDNAとストリンジェントな条件下でハイブリダイズし、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質をコードするDNA、
を挙げることができる。
本発明の形質転換体としては、上記2のDNAを含む組換え体DNAを用い、宿主細胞を公知の方法で形質転換して得られる形質転換体を挙げることができ、宿主細胞としては、細菌、酵母、動物細胞、昆虫細胞および植物細胞、好ましくは細菌、より好ましくは原核細胞、より好ましくはエシェリヒア(Escherichia)属に属する微生物を挙げることができる。
本発明のDNAは、例えば、配列番号2で表される塩基配列に基づき設計することができるプローブを用い、糸状菌等の微生物、好ましくはAspergillus属、Emericella属等に属する微生物、特に好ましくはEmericella nidulans等に属する微生物により取得することができる。
更に、決定されたDNAの塩基配列に基づいて、パーセプティブ・バイオシステムズ社製8905型DNA合成装置等を用いて化学合成することにより目的とするDNAを調製することもできる。
上記のようにして取得されるDNAとして、例えば、配列番号2で表される塩基配列を有するDNAを挙げることができる。
本発明のDNAをもとにして、必要に応じて、本発明の蛋白質をコードする部分を含む適当な長さのDNA断片を調製する。また、該蛋白質をコードする部分の塩基配列を、宿主の発現に最適なコドンとなるように、塩基を置換することにより、該蛋白質の生産率が向上した形質転換体を取得することができる。
該組換え体DNAを、該発現ベクターに適合した宿主細胞に導入することにより、本発明の蛋白質を生産する形質転換体を得ることができる。
宿主細胞としては、細菌、酵母、動物細胞、昆虫細胞等、植物細胞等、目的とする遺伝子を発現できるものであればいずれも用いることができる。
発現ベクターとしては、上記宿主細胞において自律複製可能ないしは染色体中への組込が可能で、本発明のDNAを転写できる位置にプロモーターを含有しているものが用いられる。
本発明のDNAを発現ベクターに結合させた組換え体DNAにおいては、転写終結配列は必ずしも必要ではないが、構造遺伝子の直下に転写終結配列を配置することが好ましい。
このような組換え体DNAとしては、例えばpET21-plu1440を挙げることができる。
マウス・ミエローマ細胞としては、SP2/0、NSO等、ラット・ミエローマ細胞としてはYB2/0等、ヒト胎児腎臓細胞としてはHEK293(ATCC CRL-1573)、ヒト白血病細胞としてはBALL-1等、アフリカミドリザル腎臓細胞としてはCOS-1、COS-7等を挙げることができる。
即ち、組換え遺伝子導入ベクターおよびバキュロウイルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウイルスを得た後、さらに組換えウイルスを昆虫細胞に感染させ、蛋白質を生産させることができる。
バキュロウイルスとしては、例えば、夜盗蛾科昆虫に感染するウイルスであるアウトグラファ・カリフォルニカ・ヌクレアー・ポリヘドロシス・ウイルス(Autographa californica nuclear polyhedrosis virus) 等を用いることができる。
昆虫細胞としては、スポドプテラ・フルギペルダ(Spodoptera frugiperda)の卵巣細胞、トリコプルシア・ニ(Trichoplusia ni)の卵巣細胞、カイコ卵巣由来の培養細胞等を用いることができる。
スポドプテラ・フルギペルダの卵巣細胞としてはSf9、Sf21(バキュロウイルス・イクスプレッション・ベクターズ ア・ラボラトリー・マニュアル)等、トリコプルシア・ニの卵巣細胞としてはHigh 5、BTI-TN-5B1-4(インビトロジェン社製)等、カイコ卵巣由来の培養細胞としてはボンビクス・モリ(Bombyx mori)N4等を挙げることができる。
プロモーターとしては、植物細胞中で機能するものであればいずれのものを用いてもよく、例えば、カリフラワーモザイクウイルス(CaMV)の35Sプロモーター、イネアクチン1プロモーター等を挙げることができる。
宿主細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファルファ、イネ、コムギ、オオムギ等の植物細胞等を挙げることができる。
組換えベクターの植物細胞への導入方法としては、植物細胞にDNAを導入する方法であればいずれも用いることができ、例えば、アグロバクテリウム(Agrobacterium)を用いる方法(特開昭59-140885、特開昭60-70080、WO94/00977)、エレクトロポレーション法(特開昭60-251887)、パーティクルガン(遺伝子銃)を用いる方法(特許第2606856、特許第2517813)等を挙げることができる。
上記5の方法で得られる形質転換体を培地に培養し、培養物中に本発明の蛋白質を生成、蓄積させ、該培養物から採取することにより、該蛋白質を製造することができる。
本発明の蛋白質を製造するための上記形質転換体の宿主としては、細菌、酵母、動物細胞、昆虫細胞等、植物細胞等いずれであってもよいが、好ましくは細菌、より好ましくはエシェリヒア属に属する微生物、さらに好ましくはエシェリヒア・コリに属する微生物を挙げることができる。
酵母、動物細胞、昆虫細胞または植物細胞により発現させた場合には、糖あるいは糖鎖が付加された蛋白質を得ることができる。
上記形質転換体を培地に培養する方法は、宿主の培養に用いられる通常の方法に従って行うことができる。
また、培養中必要に応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に添加してもよい。
培養は、通常pH6~8、25~40℃、5%CO2存在下等の条件下で1~7日間行う。
また、培養中必要に応じて、カナマイシン、ペニシリン、ストレプトマイシン等の抗生物質を培地に添加してもよい。
培養は、通常pH6~7、25~30℃等の条件下で1~5日間行う。
また、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい。
培養は、通常pH5~9、20~40℃の条件下で3~60日間行う。
また、培養中必要に応じて、カナマイシン、ハイグロマイシン等の抗生物質を培地に添加してもよい。
すなわち、遺伝子組換えの手法を用いて、本発明の蛋白質の活性部位を含む蛋白質の手前にシグナルペプチドを付加した形で生産させることにより、該蛋白質を宿主細胞外に積極的に分泌させることができる。
動物個体の場合は、例えば、本発明のDNAまたは本発明の製造法に用いられるDNAを導入したトランスジェニック非ヒト動物を飼育し、本発明の蛋白質を該動物中に生成、蓄積させ、該動物中より該蛋白質を採取することにより、該蛋白質を製造することができる。該動物中の該蛋白質を生成、蓄積させる場所としては、例えば、該動物のミルク(特開昭63-309192)、卵等を挙げることができる。この際に用いられるプロモーターとしては、動物で機能するものであればいずれも用いることができるが、例えば、乳腺細胞特異的なプロモーターであるαカゼインプロモーター、βカゼインプロモーター、βラクトグロブリンプロモーター、ホエー酸性プロテインプロモーター等が好適に用いられる。
例えば、本発明の蛋白質が、細胞内に溶解状態で生産された場合には、培養終了後、細胞を遠心分離により回収し、水系緩衝液に懸濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノミル等により細胞を破砕し、無細胞抽出液を得る。
該可溶化液を、蛋白質変性剤を含まないあるいは蛋白質変性剤の濃度が蛋白質が変性しない程度に希薄な溶液に希釈、あるいは透析し、該蛋白質を正常な立体構造に構成させた後、上記と同様の単離精製法により精製標品を得ることができる。
即ち、該培養物を上記と同様の遠心分離等の手法により処理することにより可溶性画分を取得し、該可溶性画分から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。
このようにして取得される蛋白質として、例えば、配列番号1で表されるアミノ酸配列からなる蛋白質を挙げることができる。
本発明の蛋白質は、糖化蛋白質に蛋白質分解酵素が作用して糖化蛋白質より生成する糖化ペプチドに作用し、過酸化水素を生成する性質を有することから、各種試料中の糖化蛋白質の測定に用いることができる。具体的には、試料を蛋白質分解酵素と反応させて糖化ペプチドを生成させ、生成した糖化ペプチドと、本発明の蛋白質とを反応させ、当該糖化ペプチドと本発明の蛋白質との反応により生成する物質又は当該糖化ペプチドと本発明の蛋白質との反応において消費される物質を測定することにより、試料中の糖化蛋白質を測定することができる。試料中の糖化蛋白質の測定に係る反応は、後述の水性媒体中で行われてもよい。本発明における糖化蛋白質としては、例えばヘモグロビンA1c等の糖化ヘモグロビンや糖化アルブミン等が挙げられ、糖化ヘモグロビンが好ましく、ヘモグロビンA1cが特に好ましい。
以下、本発明の測定方法について説明する。
本発明の測定方法に用いられる試料としては、糖化蛋白質を含む試料であれば特に制限はなく、例えば全血液、血漿、血清、血球、細胞試料、尿、髄液、汗、涙液、唾液、皮膚、粘膜、毛髪等の生体試料、及び食品等が挙げられる。試料としては、全血液、血漿、血清、血球等が好ましく、全血液、血球等が特に好ましい。なお、全血液には、全血液由来の血球分画に血漿が混合している試料も含まれる。これらの試料は、溶血、分離、希釈、濃縮、精製等の前処理を施したものを用いてもよい。
即ち、精製ヘモグロビンを含む試料又は全血液を含む試料に蛋白質分解酵素を作用させると、例えばα-FVH、α-FV、ε-FK、α-FVL等が生成し、α-FVH及びα-FVLは糖化ヘモグロビンに由来し、α-FVHはヘモグロビンA1cに特異的に由来する。
従って、ヘモグロビンA1cを測定する場合は、α-FVHを特異的に測定すればよい。本発明の蛋白質は、α-FVHに対する反応性が高く、かつε-FKに対する反応性が低いため、ヘモグロビンA1cを効果的に測定することができる。
本発明に使用しうる蛋白質分解酵素は、試料中に含まれる測定すべき糖化蛋白質に作用するものであればいかなるものを用いてもよく、例えば動物、植物、微生物由来のプロテアーゼ、メタロプロテアーゼ、エンドプロテアーゼ、エキソプロテアーゼ、セリンプロテアーゼ、システインプロテアーゼ、酸性プロテアーゼ、アルカリ性プロテアーゼ、チオールプロテアーゼ等が挙げられる。
(1) バチルス(Bacillus)属由来プロテアーゼ;ズブチリシン(Subtilisin)、プロテアーゼ-タイプ-VIII、プロテアーゼ-タイプ-IX、プロテアーゼ-タイプ-X、プロテアーゼ-タイプ-XV、プロテアーゼ-タイプ-XXIV、プロテアーゼ-タイプ-XXVII、プロテアーゼ-タイプ-XXXI、プロテイナーゼTypeVII、バチルスリケニフォルミス由来プロテアーゼ(以上、シグマ社製)、サーモリシン(和光純薬工業社製)、オリエンターゼ-90N、オリエンターゼ-10NL、オリエンターゼ-22BF、オリエンターゼ-Y、オリエンターゼ-5BL、ヌクレイシン(以上、エイチビィアイ株式会社製)、プロレザーFG-F、プロテアーゼNL「アマノ」、プロテアーゼS「アマノ」G、プロテアーゼN「アマノ」G(以上、天野エンザイム社製)、GODO-BNP、GODO-BAP、GODO高純度プロテアーゼ(以上、合同酒清社精製)、プロチン-AC10F、プロチン-NL10、プロチン-NC25、プロチン-NY10、プロチン-PC10F、プロチン-PS10、デスキン、デピレイス、ビオソーク、サモアーゼ-PC10F、サーモリシン(以上、大和化成社製)、トヨチームNEP、中性プロテアーゼ(以上、東洋紡績社製)、ニュートラーゼ、エスペラーゼ、サビナーゼ、デュラザイム、バイオフィードプロ、アルカラーゼ、NUE、ピラーゼ、クリアーレンズプロ、エバラーゼ、ノボザイム-FM、ボラン(以上、ノボノルディスクバイオインダストリー社製)、エンチロン-NBS、エンチロン-SA(以上、洛東化成工業社製)、ナガーゼ(Nagarse)、ビオプラーゼAPL-30、ビオプラーゼSP-4FG、ビオプラーゼXL-416F、ビオプラーゼAL-15FG、ペクチナーゼXP-534(以上、ナガセケムテックス社製)、アロアーゼAP-10、プロテアーゼYB、(以上、ヤクルト薬品工業社製)、コロラーゼ-N、コロラーゼ-7089、ベロンW(以上、樋口商会社製)、キラザイム P-1、ディスパーゼ(以上、ロシュ社製)、サチライシン(ベーリンガー・マンハイム社製)、プロテイナーゼN、プロテイナーゼBacterial Subtilisin(以上、フルカ社製)、プロナーゼE(科研製薬社製)等。
メタロプロテアーゼとしては、例えばサーモリシン、プロテアーゼN等が挙げられる。エンドプロテアーゼとしては、例えばサーモリシン、パパイン、ズブチリシン、ペプシン、トリプシン、キモトリプシン等が挙げられる。エキソプロテアーゼとしては、例えばアミノペプチダーゼ、カルボキシペプチダーゼ等が挙げられる。セリンプロテアーゼとしては、サーミターゼ、プロテイナーゼK、トリプシン、キモトリプシン、トロンビン、プラスミン、エラスターゼ等が挙げられる。システインプロテアーゼとしては、パパイン、カスパーゼ等が挙げられる。酸性プロテアーゼとしては、ペプシン、カテプシンD等が挙げられる。アルカリ性プロテアーゼとしては、オリエンターゼ22BF等が挙げられる。チオールプロテアーゼは、例えばパパイン、フィシン、ブロメライン等が挙げられる。
本発明の試料中の測定すべき糖化蛋白質の測定は、下記(i)~(iii)の工程を順次行うことによって行うことができる。
(i)試料を蛋白質分解酵素と反応させて糖化ペプチドを生成させる工程、
(ii)生成した糖化ペプチドを本発明の蛋白質と反応させる工程、及び、
(iii)(ii)の工程で生成した物質、又は、消費された物質を測定する工程。
各工程の反応の反応温度としては、例えば10~50℃で、好ましくは20~40℃であり、反応時間としては、1秒間~60分間、好ましくは1~10分間である。
過酸化水素測定試薬は、生成した過酸化水素を検出可能な物質へ変換するための試薬である。検出可能な物質としては、例えば色素、光等が挙げられるが、色素が好ましい。
測定すべき糖化蛋白質を含む試料は、必要に応じて生体試料から分離することができる。分離方法としては、遠心、濾過、血球分離膜を用いた方法等が挙げられる。例えば遠心による分離方法は、全血液を血球、血漿もしくは血清に分離することができる。必要に応じて、該血球を生理食塩水等の等張液で洗浄することで、血漿由来の成分を除去した洗浄血球を得ることもできる。
健常人及び糖尿病患者から血液を採取、転倒混和後、25℃で5分間、遠心分離(3,000 rpm)を行う。遠心分離後、上澄みの血漿は除去する。下層部分の血球層1容に対して、4容の生理食塩水を追加し、転倒混和後、25℃で5分間、遠心分離(3,000 rpm)を行う。遠心分離後、上澄みの生理食塩水を除去する。この洗浄操作を3回繰り返した後、洗浄された血球層1容に対して9容の蒸留水を添加し、洗浄血球を得ることができる。
本発明の糖化蛋白質測定用試薬及び測定用キットは、本発明の糖化蛋白質の測定方法に使用することができる。本発明の糖化蛋白質測定用試薬は、保存、運搬、流通に適した形態として、キットの形態をとり得る。キットの形態としては、2試薬系、3試薬系等が挙げられる。
・キット1(2試薬系キット)
以下の2つの試薬を含むキット。
(1)蛋白質分解酵素を含む試薬;
(2)本発明の蛋白質を含む試薬。
以下の2つの試薬を含むキット。
(1)蛋白質分解酵素を含む試薬;
(2)本発明の蛋白質、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
以下の2つの試薬を含むキット。
(1)蛋白質分解酵素、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;
(2)本発明の蛋白質を含む試薬。
以下の2つの試薬を含むキット。
(1)蛋白質分解酵素、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;
(2)本発明の蛋白質、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
以下の3つの試薬を含むキット。
(1)蛋白質分解酵素を含む試薬;
(2)本発明の蛋白質を含む試薬;及び、
(3)本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
以下の3つの試薬を含むキット。
(1)蛋白質分解酵素、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;
(2)本発明の蛋白質を含む試薬;及び、
(3)本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
以下の3つの試薬を含むキット。
(1)蛋白質分解酵素を含む試薬;
(2)本発明の蛋白質、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;及び、
(3)本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
以下の3つの試薬を含むキット。
(1)蛋白質分解酵素、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;
(2)本発明の蛋白質、及び、本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬;及び、
(3)本発明の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む試薬。
本発明の測定用試薬及び測定用キットには、必要に応じて、それぞれ緩衝剤、安定化剤、防腐剤、影響物質除去剤、非特異反応抑制剤、界面活性剤等が含有されてもよい。緩衝剤としては、例えば前述の緩衝剤等が挙げられる。安定化剤としては、例えばエチレンジアミン四酢酸(EDTA)、シュークロース、塩化カルシウム、アミノ酸類、アルブミン、デキストラン、酢酸カルシウム等の塩類等が挙げられる。防腐剤としては、例えばアジ化ナトリウム、抗生物質等が挙げられる。影響物質除去剤としては、例えばアスコルビン酸の影響を消去するためのアスコルビン酸オキシダーゼ等が挙げられる。非特異反応抑制剤としては、デキストラン硫酸等の高分子化合物等が挙げられる。界面活性剤としては、例えば非イオン性界面活性剤、陽イオン性界面活性剤、陰イオン性界面活性剤、両性界面活性剤等が挙げられる。
本発明の測定キットにおける本発明の蛋白質の含量としては、水性媒体で溶解された状態での濃度が0.01~10,000 U/mLとなる含量が好ましく、0.1~1,000 U/mLとなる含量がより好ましい。
過酸化水素測定試薬としてパーオキシダーゼと酸化カップリング型色原体を含む試薬を使用する場合のキットにおけるパーオキシダーゼ及び該酸化カップリング型色原体の含量としては、それぞれ、水性媒体で溶解された状態での濃度が1~600 U/mL、0.5~40 g/Lとなる含量が好ましく、2~150 U/mL、1~20 g/Lとなる含量がより好ましい。
α-FVHに対する活性が比較的高く、かつε-FKに対する活性が低い酵素を生産する菌として、Emericella nidulans KY125株を選択した。
次に、E. nidulansの類縁の菌で、かつその全ゲノム配列が解読されているAspergillus nidulans FGSC A4株のDNA配列(Nature, 438, 1105 (2005))を参考にしたプライマー(配列番号5、配列番号6)を用いて、フルクトシルペプチドオキシダーゼ遺伝子の全長をPCR法を用いて増幅した。そのDNA配列をDNA シークエンサーを用いて解読したところ、KY125株のフルクトシルペプチドオキシダーゼ遺伝子は、6個のエキソンの間に、5個のイントロンを含んでいた。そこで、オーバーラップPCR法(Nucleic Acids Res., 16, 7351 (1988))によってエキソン部分のみを増幅し、それらを結合し、エキソンのみからなる成熟型フルクトシルペプチドオキシダーゼ遺伝子を造成した。そしてそのDNA配列を解読したところ、成熟型のフルクトシルペプチドオキシダーゼ遺伝子は、1317 bp、438アミノ酸からなることが判明した。
実施例1で得られたフルクトシルペプチドオキシダーゼ遺伝子に対してランダム変異を導入した。ランダム変異の導入は、Stratagene社の「GeneMorph II Random Mutagenesis Kit」を用いて行った。pTrcFPOX-1をテンプレートDNAとし、そのフルクトシルペプチドオキシダーゼ遺伝子の5’-側上流と3’-側下流に相当する領域に対応するプライマー(配列番号5、配列番号6)を用いるPCR法により、塩基置換の導入と全長の増幅を同時に行った。
実施例2で得られたFPOX-9を保有するE. coli XL1-Blue株をアンピシリン50 mg/Lを含有するLB培地を10 mL含む試験管10本に植菌し、30℃で、24時間振とう培養した。これらの培養液をアンピシリン50 mg/LとIPTG20 mg/Lを含有するLB培地を300 mL含む三角フラスコ10個にそれぞれ移しかえ、30℃で、24時間振とう培養した。
実施例2で得られた新規FPOX-9の糖化ペプチド及び/又は糖化アミノ酸への基質特異性を、以下の第1試薬~第3試薬を用いて測定した。
トリス緩衝液(pH7.5) 100 mmol/L
トリス緩衝液(pH7.5) 100 mmol/L
フルクトシルペプチドオキシダーゼFPOX-9 2μL
4-アミノアンチピリン 0.5 mmol/L
TOOS 0.2 mmol/L
パーオキシダーゼ(西洋わさび由来) 10 U/mL
ここで、FPOX-9溶液は、実施例3で調製したものを使用した。
トリス緩衝液(pH7.5) 100 mmol/L
α-FVH,α-FV又はε-FK X mmol/L
(α-FVH又はα-FVを用いた場合は、X=0,0.05,0.1,0.5,1,2,5,10)
(ε-FKを用いた場合は、X=0,1,2,5,10,20,40, 50, 80, 100)
ΔA0=A0 x- A0’(Abs) (I)
ΔAs=As x- As’(Abs) (II)
酵素活性(U/mL)={(ΔAs―ΔA0)×総反応液量(mL)}/{分子吸光係数ε/1000×酵素液量(mL)×0.5×反応時間(min)×光路長(cm)} (III)
pTrcFPOX-9をテンプレートDNAとし、そのフルクトシルペプチドオキシダーゼ遺伝子の5’-側上流と3’-側下流に相当する領域に対応するプライマー(配列番号5、配列番号6)を用いて、「GeneMorph II Random Mutagenesis Kit」を用いるPCR法により、塩基置換の導入と全長の増幅を同時に行った。
実施例2で得られたFPOX-15の糖化ペプチド及び/又は糖化アミノ酸への基質特異性を、以下の第1試薬~第3試薬を用いて測定した。
リン酸二水素ナトリウム(pH8.0) 100 mmol/L
フルクトシルペプチドオキシダーゼFPOX-15 2μL
ここで、FPOX-15溶液は、実施例3で得られた溶液を4倍希釈したものを用いた。
リン酸二水素ナトリウム(pH6.0またはpH7.0またはpH8.0) 100mmol/L
パーオキシダーゼ(西洋わさび由来) 3U/mL
4-アミノアンチピリン 0.5mmol/L
EMSE 0.4mmol/L
α-FVH、α-FV又はε-FK X mmol/L
(α-FVH又はα-FVを用いた場合は、X=0, 2, 2.5, 3, 3.66)
(ε-FKを用いた場合は、X=0, 20, 30, 40, 50)
ΔA0=A0 X-A0’(Abs) (I)
ΔAS=AS X-AS’(Abs) (II)
酵素活性(U/mL)={(ΔAS―ΔA0)×総反応液量(mL)}/{分子吸光係数ε/1000×酵素液量(mL)×0.5×反応時間(min)×光路長(cm)} (III)
FPOX-9及びFPOX-15の1 mg/mLリン酸緩衝液(pH7.0)溶液の各溶液(3μL)を、Phast System(全自動電気泳動システム、GE Healthcare社)の等電点電気泳動ゲルにアプライし、操作手順に従って、電気泳動、染色、脱色操作を行い、各フルクトシルペプチドオキシダーゼのpIを決定した。その結果を表6に示す。
フルクトシルペプチドオキシダーゼFPOX-9及びFPOX-15の安定性に対するpHの影響を、以下の第1試薬~第3試薬を用いて評価した。評価に際して、第1試薬として、調製直後の試薬、調製後30℃にて24時間(1日)保存した試薬、調製後30℃にて5日間保存した試薬を用いた。
Bis-Tris(pH5.0またはpH6.0またはpH7.0またはpH8.0) 100 mmol/L
フルクトシルペプチドオキシダーゼFPOX-9またはFPOX-15 2μL
ここで、FPOX-9溶液は、実施例3で得られた溶液を20倍希釈したもの、FPOX-15溶液は、実施例3で得られた溶液を20倍希釈したものを用いた。
リン酸二水素ナトリウム(pH6.0またはpH7.0またはpH8.0) 100 mmol/L
パーオキシダーゼ(西洋わさび由来) 3 U/mL
4-アミノアンチピリン 0.5 mmol/L
EMSE 0.4 mmol/L
α-FG(フルクトシルグリシン) 0または15 mmol/L
ΔA0=A0 X-A0’(Abs) (I)
ΔAS=AS X-AS’(Abs) (II)
酵素活性(U/mL)={(ΔAS―ΔA0)×総反応液量(mL)}/{分子吸光係数ε/1000×酵素液量(mL)×0.5×反応時間(min)×光路長(cm)} (III)
酵素活性残存率E'(%)=(E1day、又は、E5day)/E0day×100 (IV)
フルクトシルペプチドオキシダーゼFPOX-9及びFPOX-15の安定性に対する金属の影響を、以下の第1試薬~第3試薬を用いて評価した。評価に際して、第1試薬として、調製直後の試薬、調製後5℃にて24時間(1日)保存した試薬、調製後30℃にて24時間(1日)保存した試薬を用いた。
Bis-Tris(pH7.0) 100 mmol/L
フルクトシルペプチドオキシダーゼFPOX-9またはFPOX-15 2μL
金属イオン X mmol/L
(金属イオンがNa+、K+、Li+の場合:X=100;金属イオンがMg2+、Ca2+の場合:X=10;金属イオンがCr3+、Mn2+、Fe3+、Co2+、Ni2+、Cu2+、Zn2+、Ag+、Cd2+、Pb2+、Ba2+、Al3+、Sr2+の場合:X=0.1)
リン酸二水素ナトリウム(pH8.0) 100 mmol/L
パーオキシダーゼ(西洋わさび由来) 3 U/mL
4-アミノアンチピリン 0.5 mmol/L
EMSE 0.4 mmol/L
α-FG(フルクトシルグリシン) 0または15 mmol/L
ΔA0=A0 X-A0’(Abs) (I)
ΔAS=AS X-AS’(Abs) (II)
酵素活性(U/mL)={(ΔAS―ΔA0)×総反応液量(mL)}/{分子吸光係数ε/1000×酵素液量(mL)×0.5×反応時間(min)×光路長(cm)} (III)
酵素活性残存率E''(%)=(E’1day/E’0day)×100 (V)
配列番号6-人工配列の説明:合成DNA
Claims (17)
- 以下の[1]~[4]のいずれかに記載の蛋白質。
[1]配列番号1で表されるアミノ酸配列を有する蛋白質
[2]配列番号1で表されるアミノ酸配列において、1以上のアミノ酸が欠失、置換または付加したアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質
[3]配列番号1で表されるアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質
[4]寄託番号FERM BP-11026として寄託された大腸菌XL1-Blue MRF'株が担持する発現プラスミドによってコードされるフルクトシルペプチドオキシダーゼ活性を有する蛋白質 - 配列番号3で表されるアミノ酸配列からなる、請求項1に記載の蛋白質。
- 以下の[1]~[3]のいずれかに記載のDNA。
[1]請求項1記載の蛋白質をコードするDNA
[2]配列番号2で表される塩基配列を有するDNA
[3]配列番号2で表される塩基配列と相補的な塩基配列を有するDNAとストリンジェントな条件下でハイブリダイズし、かつフルクトシルペプチドオキシダーゼ活性を有する蛋白質をコードするDNA - 配列番号3で表されるアミノ酸配列からなる蛋白質をコードする、請求項3に記載のDNA。
- 配列番号4で表される塩基配列からなる、請求項3に記載のDNA。
- 請求項3~5のいずれかに記載のDNAを含有する組換え体DNA。
- 請求項6記載の組換え体DNAを有する形質転換体。
- 請求項7記載の形質転換体を培地に培養し、培養物中に該蛋白質を生成、蓄積させ、該培養物より該蛋白質を採取する請求項1又は2記載の蛋白質の製造方法。
- 試料を蛋白質分解酵素と反応させて糖化ペプチドを生成させ、次いで、生成した糖化ペプチドを請求項1又は2記載の蛋白質と反応させ、当該糖化ペプチドと当該蛋白質との反応により生成した物質又は当該糖化ペプチドと当該蛋白質との反応において消費された物質を測定することを特徴とする、試料中の糖化蛋白質の測定方法。
- 糖化蛋白質が、糖化ヘモグロビンである請求項9記載の測定方法。
- 糖化ヘモグロビンが、ヘモグロビンA1cである請求項10記載の測定方法。
- 蛋白質分解酵素、請求項1又は2記載の蛋白質を含む糖化蛋白質測定用試薬。
- さらに、請求項1又は2記載の蛋白質と糖化蛋白質から生成する糖化ペプチドとの反応により生成する生成物を測定するための試薬を含む請求項12記載の試薬。
- 生成物が、過酸化水素である請求項13記載の試薬。
- 糖化蛋白質が、糖化ヘモグロビンである請求項12~14のいずれかに記載の試薬。
- 糖化ヘモグロビンが、ヘモグロビンA1cである請求項15記載の試薬。
- 寄託番号FERM BP-11026として寄託された大腸菌XL1-Blue MRF'株。
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2739881A CA2739881A1 (en) | 2008-10-09 | 2009-10-08 | Novel fructosyl peptide oxidase |
US13/123,049 US8304249B2 (en) | 2008-10-09 | 2009-10-08 | Fructosyl peptide oxidase |
JP2010532958A JP5665081B2 (ja) | 2008-10-09 | 2009-10-08 | 新規フルクトシルペプチドオキシダーゼ |
EP09819249.5A EP2357228B1 (en) | 2008-10-09 | 2009-10-08 | Novel fructosyl peptide oxidase |
CN200980148386.8A CN102232111B (zh) | 2008-10-09 | 2009-10-08 | 果糖基肽氧化酶 |
HK12103960.6A HK1163734A1 (en) | 2008-10-09 | 2012-04-20 | Fructosyl peptide oxidase |
US13/633,410 US8790905B2 (en) | 2008-10-09 | 2012-10-02 | Fructosyl peptide oxidase |
US14/267,729 US8883142B2 (en) | 2008-10-09 | 2014-05-01 | Fructosyl peptide oxidase |
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EP (1) | EP2357228B1 (ja) |
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KR (1) | KR20110069138A (ja) |
CN (1) | CN102232111B (ja) |
CA (1) | CA2739881A1 (ja) |
HK (1) | HK1163734A1 (ja) |
WO (1) | WO2010041715A1 (ja) |
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JP2015037402A (ja) | 2015-02-26 |
US20120003678A1 (en) | 2012-01-05 |
HK1163734A1 (en) | 2012-09-14 |
EP2357228B1 (en) | 2017-05-10 |
US8304249B2 (en) | 2012-11-06 |
EP2357228A4 (en) | 2012-04-25 |
CN102232111A (zh) | 2011-11-02 |
US8883142B2 (en) | 2014-11-11 |
EP2357228A1 (en) | 2011-08-17 |
US20140057333A1 (en) | 2014-02-27 |
JPWO2010041715A1 (ja) | 2012-03-08 |
JP5665081B2 (ja) | 2015-02-04 |
KR20110069138A (ko) | 2011-06-22 |
CN102232111B (zh) | 2014-12-03 |
CA2739881A1 (en) | 2010-04-15 |
US20140234886A1 (en) | 2014-08-21 |
US8790905B2 (en) | 2014-07-29 |
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