JPH09187280A - Use of dna having gene information on pyruvate oxidase - Google Patents

Abstract

PURPOSE: To obtain a pyruvate oxidase for research and diagnosis by incorporating a pyruvate oxidase gene coding a specific amino acid sequence into an expression vector and transfecting a host microorganism with the expression vector to give a transformant and expressing this gene information. CONSTITUTION: A polydeoxyribonucleic acid as a pyruvate oxidase gene coding an amino acid sequence from the N-terminus of the formula (A is an amino acid residue, H, acetyl; B is an amino acid residue, OH, NH2 ) or a polydeoxyribonucleic acid containing this gene as a constitution component is incorporated into an expression vector and a host microorganism is transfected with the recombinant DNA. Then, the transformant is cultured to express the gene information of this polydeoxyribonucleic acid, and a polypeptide as a pyruvate oxidase or a polypeptide including the pyruvate oxidase as a constitution component is collected whereby the objective pyruvate oxidase is efficiently obtained.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a pyruvate oxidase obtained by expressing the genetic information of polydeoxyribonucleic acid by a transformant carrying a polydeoxyribonucleic acid having the genetic information of the novel pyruvate oxidase. It relates to a manufacturing method.

[0002]

Pyruvate oxidase catalyzes the reaction of pyruvate, phosphate and oxygen to produce acetyl phosphate, carbon dioxide and hydrogen peroxide.
Lactobacillus delb
rucki) [Williams, FR & Hager, LP (1966) Arch.
Biochem.Biophys. 116 168-176] Pediococcus, Streptococcu
s), Aerococcus virid
ans) [Japanese Patent Publication No. 58-40465].

Further, since pyruvate oxidase is an oxidase using pyruvate as a substrate, it is not only used for the quantification of pyruvate present in body fluids such as serum, but also glutamic acid-oxaloacetate transaminase,
By measuring the amount of pyruvate produced in the pyruvate production system such as glutamate-pyruvate transaminase, lactate dehydrogenase, neuraminidase-N acetylneuraminate aldolase, various substances that are substrates of enzymes involved in the pyruvate production system It is extremely useful as a reagent for research and a reagent for clinical diagnosis such as quantification or measurement of each enzyme activity involved in the pyruvate generation system.

[0004]

The pyruvate oxidase-producing bacterium that has been reported hitherto has not only the disadvantage that the productivity of pyruvate oxidase is low and the manufacturing cost is high, but also the coexisting enzyme of other species. Is very difficult to remove, and the purification cost is very high in order to obtain high-purity, high-quality pyruvate oxidase, and it is not always satisfactory to be widely used as a research reagent or clinical diagnostic reagent. Was not.

Further, the detailed chemical structure of pyruvate oxidase, which catalyzes the previously reported reaction of producing acetyl phosphate, carbon dioxide and hydrogen peroxide from pyruvic acid, phosphoric acid and oxygen, has not been reported.

[Means for Solving the Problems] The inventors of the present invention have made diligent studies in order to improve the productivity of the pyruvate oxidase, and succeeded in collecting the pyruvate oxidase gene and analyzing the primary structure of the gene. As described below, a manufacturing method with high productivity was established by applying the dynamic method.

[0006] That is, the present invention provides a polydeoxyribonucleic acid, which is a pyruvate oxidase gene encoding the following amino acid sequence from the N-terminal side, or a recombinant DNA in which an expression vector is incorporated with polydeoxyribonucleic acid having the gene as a constituent. To a host microorganism to obtain a transformant, the transformant is cultured to express the genetic information of the polydeoxyribonucleic acid, and then the polypeptide which is pyruvate oxidase or the pyruvate oxidase is a constituent component. This is a method for producing pyruvate oxidase by collecting a polypeptide. A-Ser Asp Asn Lys Ile Asn Ile Gly Leu Ala Val Met Lys Ile Leu Glu Ser Trp Gly Ala Asp Thr Ile Tyr Gly Ile Pro Ser Gly Thr Leu Ser Ser Leu Met Asp Ala Met Gly Glu Glu Glu Aslu Asn Val Lys Phe Leu Gln Val Lys His Glu Glu Val Gly Ala Met Ala Ala Val Met Gln Ser Lys Phe Gly Gly Asn Leu Gly Val Thr Val Gly Ser Gly Gly Pro Gly Ala Ser His Leu Ile Asn Gly Leu Tyr Asp Ala Ala Met Asp Asn Ile Pro Val Val Ala Ile Leu Gly Ser Arg Pro Gln Arg Glu Leu Asn Met Asp Ala Phe Gln Glu Leu Asn Gln Asn Pro Met Tyr Asp His Ile Ala Val Tyr Asn Arg Arg Val Ala Tyr Ala Glu Gln Leu Pro Lys Leu Val Asp Glu Ala Ala Arg Met Ala Ile Ala Lys Arg Gly Val Ala Val Leu Glu Val Pro Gly Asp Phe Ala Lys Val Glu Ile Asp Asn Asp Gln Trp Tyr Ser Ser Ala Asn Ser Leu Arg Lys Tyr Ala Pro Ile Ala Pro Ala Ala Gln Asp Ile Asp Ala Ala Val Glu Leu Leu Asn Asn Ser Lys Arg Pro Val Ile Tyr Ala Gly Ile Gly Thr Met Gly His Gly Pro Ala Val Gln Glu Leu Ala Arg Lys Ile Lys Ala Pro Val Ile Thr Thr Gly Lys Asn Phe Glu Thr Phe Glu Trp Asp Ph e Glu Ala Leu Thr Gly Ser Thr Tyr Arg Val Gly Trp Lys Pro Ala Asn Glu Thr Ile Leu Glu Ala Asp Thr Val Leu Phe Ala Gly Ser Asn Phe Pro Phe Ser Glu Val Glu Gly Thr Phe Arg Asn Val Asp Asn Phe Ile Gln Ile Asp Ile Asp Pro Ala Met Leu Gly Lys Arg His His Ala Asp Val Ala Ile Leu Gly Asp Ala Gly Leu Ala Ile Asp Glu Ile Leu Asn Lys Val Asp Ala Val Glu Glu Ser Ala Trp Trp Thr Ala Asn Leu Lys Asn Ile Ala Asn Trp Arg Glu Tyr Ile Asn Met Leu Glu Thr Lys Glu Glu Gly Asp Leu Gln Phe Tyr Gln Val Tyr Asn Ala Ile Asn Asn His Ala Asp Glu Asp Ala Ile Tyr Ser Ile Asp Val Gly Asn Ser Thr Gln Thr Ser Ile Arg His Leu His Met Thr Pro Lys Asn Met Trp Arg Thr Ser Pro Leu Phe Ala Thr Met Gly Ile Ala Ile Pro Gly Gly Leu Gly Ala Lys Asn Thr Tyr Pro Asp Arg Gln Val Trp Asn Ile Ile Gly Asp Gly Ala Phe Ser Met Thr Tyr Pro Asp Val Val Thr Asn Val Arg Tyr Asn Met Pro Val Ile Asn Val Val Phe Ser Asn Thr Glu Tyr Ala Phe Ile Lys Asn Lys Tyr Glu Asp Thr Asn Lys Asn Leu Phe Gly Val Asp Phe Thr Asp Val Asp Tyr Ala Lys Ile Al a Glu Ala Gln Gly Ala Lys Gly Phe Thr Val Ser Arg Ile Glu Asp Met Asp Arg Val Met Ala Glu Ala Val Ala Ala Asn Lys Ala Gly His Thr Val Val Ile Asp Cys Lys Ile Thr Gln Asp Arg Pro Ile Pro Val Glu Thr Leu Lys Leu Asp Ser Lys Leu Tyr Ser Glu Asp Glu Ile Lys Ala Tyr Lys Glu Arg Tyr Glu Ala Ala Asn Leu Val Pro Phe Arg Glu Tyr Leu Glu Ala Glu Gly Leu Glu Ser Lys Tyr Ile Lys −B (I) in, a is an amino acid residue, a hydrogen atom or an acetyl group, B is related amino acid residue, to an -OH or -NH _2].

In the above polypeptide (I), the amino acid residue represented by A is composed of one or a plurality of amino acid residues, and A is preferably a hydrogen atom, Met or a signal peptide. The compound represented by B may be an acid amide or one or more amino acid residues. Pyruvate,
The polydeoxyribonucleic acid that is a pyruvate oxidase gene that catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from phosphate and oxygen, or the polydeoxyribonucleic acid that is a component of the gene, is at least pyruvic acid, phosphate, and oxygen. Any polydeoxyribonucleic acid containing the pyruvate oxidase gene itself which catalyzes the reaction that produces acetyl phosphate, carbon dioxide and hydrogen peroxide from A polydeoxyribonucleic acid, which is a base sequence encoding the amino acid sequence of a polypeptide consisting of pyruvate oxidase that catalyzes the reaction of producing pyruvic acid, phosphoric acid and oxygen from acetyl phosphate, carbon dioxide and hydrogen peroxide Can be done.

Further, in the amino acid sequence of the polypeptide which is pyruvate oxidase represented by the formula (II),
Any one of a series of codons corresponding to each amino acid
The polydeoxyribonucleic acid may be any polydeoxyribonucleic acid, and at the 5'end of the polydeoxyribonucleic acid one or more codons other than the nonsense codon and / or 3 '
It may be a polydeoxyribonucleic acid having at least one codon at the end. For example, as a typical example, the formula from the 5 ′ end side [In the formula, X represents a codon or hydrogen atom other than TAA, TAG and TGA, and Y represents a codon or hydrogen atom].

With respect to the base sequence represented by the formula (III),
The codon represented by X may be any codon that encodes an amino acid, and may further have one or more codons that encode an amino acid at the 5′-terminal side thereof, but preferably in the ATG or signal peptide. Mention may be made of the corresponding polydeoxyribonucleic acids. The codon represented by Y may be any translation termination codon or a codon that encodes an amino acid, and may further have one or more codons that encode an amino acid at the 3'-terminal side. It is preferable to have a translation stop codon at the 3'end of the plurality of codons.

Polydeoxyribonucleic acid having a pyruvate oxidase gene as a constituent, or polydeoxyribonucleic acid having a nucleotide sequence encoding the amino acid sequence represented by formula (II) or formula (III)
The polydeoxyribonucleic acid represented by, for example, is obtained by isolating and purifying the DNA of a microorganism that is a donor of the pyruvate oxidase gene that produces pyruvate oxidase, and then cleaving it with DNA using ultrasonic waves, restriction enzymes, etc. A linear expression vector is ligated and ligated at the blunt or sticky ends of both DNAs with DNA ligase or the like, and the recombinant DNA vector thus obtained is transferred to a replicable host microorganism. Then, the vector marker and pyruvate oxidase Microorganisms retaining the recombinant DNA vector obtained by screening with activity as an index are cultured, the recombinant DNA vector is separated and purified from the cultured cells, and then the recombinant DNA vector is treated with a pyruvate oxidase gene. It is sufficient to collect a certain polydeoxyribonucleic acid.

The pyruvate oxidase gene donor may be any microorganism capable of producing pyruvate oxidase, for example, Japanese Patent Laid-Open No. 54-12.
6791, JP-A-59-159777, JP-A-59-162877, Arch.Biochem.Biophy
s. 116 168-176 (1966), Lactobacillus delbrucki,
Pediococcus sp, Streptococcus sp, Aerococcus viridans, Lactobacillus Plantaru
m), Lactobacillus
Microorganisms such as Lactobacillusaceae or Streptococcusaceae such as Salivarius) and Leuconostoc Mesenteroides are appropriately selected.

[0012] Further, a transgenic microorganism having a pyruvate oxidase-producing ability imparted by making full use of gene recombination technology may be used as a donor of the pyruvate oxidase gene. DNA derived from a microorganism that is a gene donor is collected as follows. That is, any of the above-mentioned bacteria as a donor microorganism is cultured in a liquid medium for about 1 to 3 days under aeration and agitation, and the resulting culture is collected by centrifugation and then lysed. A lysate containing the acid oxidase gene can be prepared. Lysis methods include lysozyme and β
-Glucanase and other cell wall lysing enzymes are used, and if necessary, other enzymes such as protease and surfactants such as sodium lauryl sulfate are used together. You may perform it in combination with the lysis method of.

From the lysate thus obtained, DNA
Can be isolated and purified by a conventional method, for example, by appropriately combining methods such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation and centrifugation. The method of cleaving the separated and purified microbial DNA can be carried out, for example, by ultrasonic treatment or restriction enzyme treatment.
In order to facilitate the ligation of the resulting DNA fragment with the vector, it acts on a restriction enzyme, especially a specific nucleotide sequence, for example, EcoRI, HindIII, Bam.
Type II restriction enzymes such as HI are suitable.

Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. Examples of phages include Escherichia coli
Λgt · λC, λgt · λB
Etc. can be used. The plasmid may be, for example, p when Escherichia coli is used as the host microorganism.
BR322, pBR325, pACYC184, pUC
12, pUC13, pUC18, pUC19, etc. can use pUB110, pC194, etc. when Bacillus subtillis is used as the host microorganism, and further, for Escherichia coli and Saccharomyces cerevisiae etc. It is also possible to use a shuttle vector capable of autonomously growing in two or more host microorganisms spread over it. Such a vector is prepared by using the above-mentioned microorganism D which is a pyruvate oxidase gene donor.
It is preferable to obtain a vector fragment by cleaving with the same restriction enzyme as that used for cleaving NA.

The method of ligating the microbial DNA fragment and the vector fragment may be a method using a known DNA ligase, for example, after annealing the cohesive ends of the microbial DNA fragment and the cohesive end of the vector fragment, a suitable method is used. DN
By the action of A ligase, a recombinant DNA of a microbial DNA fragment and a vector fragment is prepared. If necessary, it is also possible to transfer to a host microorganism after annealing and use an in vivo DNA ligase to prepare a recombinant DNA.

[0016] The host microorganism may be any as long as the recombinant DNA can be stably and autonomously propagated and the trait of the foreign DNA can be expressed. For example, when the host microorganism is Escherichia coli, Escherichia coli Stiffness DH1,
Escherichia coli HB101, Escherichia coli W
3110, Escherichia coli C600, etc. can be used.

As a method for transferring the recombinant DNA into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA is transferred in the presence of calcium ion and belongs to the genus Bacillus. In the case of microorganisms, the competent cell method or the protoplast method can be adopted, and the microinjection method may be used. It was found that the thus obtained transformant microorganism can stably produce a large amount of pyruvate oxidase by culturing in a nutrient medium.

The selection of the presence or absence of transfer of the target recombinant DNA into the host microorganism may be carried out by searching for a microorganism capable of simultaneously expressing the drug resistance marker of the vector carrying the target recombinant DNA and pyruvate oxidase. A microorganism that grows in a selection medium based on a drug resistance marker and that produces pyruvate oxidase may be selected. Recombinant DNA carrying the pyruvate oxidase gene once selected in this manner is removed from the transformed microorganism. Also, it can be easily carried out by transferring to another host microorganism. Further, the recombinant DNA having the pyruvate oxidase gene is cleaved with a restriction enzyme or the like to excise the DNA which is the pyruvate oxidase gene, and the vector fragment obtained by the cleavage in the same manner as this is ligated to the host. Transfer to microorganisms is also easy.

The DNA of pyruvate oxidase mutein, which is essentially pyruvate oxidase activity, is an artificial mutant gene prepared from the pyruvate oxidase gene of the present invention by a genetic engineering method. Amplified using various methods,
Finally, a pyruvate oxidase mutein can be produced by inserting this mutant gene into a vector to prepare a recombinant DNA and transferring it into a host microorganism.

Further, the base sequence of the pyruvate oxidase gene obtained by the above method is Science 214 12
It was decoded by the dideoxy method shown in 05-1210 (1981), and the amino acid sequence of pyruvate oxidase was determined from the nucleotide sequence. On the other hand, the partial amino acid sequence constituting the N-terminal part of the peptide which is pyruvate oxidase was determined as follows. That is, a pyruvate oxidase gene-donating microorganism capable of producing pyruvate oxidase is cultured in a nutrient medium to produce and accumulate pyruvate oxidase in the cells, and after the culture is completed, the resulting culture is filtered or centrifuged. Then, the bacterial cells are collected by means of a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or a suitable surfactant is added to prepare pyruvate oxidase. It is solubilized and separated and collected as an aqueous solution. The aqueous solution of pyruvate oxidase thus obtained is concentrated, or the ammonium sulfate fractionation without concentration,
High-purity pyruvate oxidase is obtained by treatment with gel filtration, adsorption chromatography, and ion exchange chromatography, and a liquid-phase protein sequencer (Beckman: B
ECKMAN System 890ME) was used to determine the partial amino acid sequence constituting the N-terminal part of the peptide that is pyruvate oxidase, and at least the partial amino acid sequence was identical to the N-terminal partial amino acid sequence of pyruvate oxidase obtained by genetic engineering. I confirmed that it is something to do.

The culture form of the host microorganism that is a transformant may be selected by considering culture conditions in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is used, or industrially deep aeration. It is advantageous to carry out agitation culture. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source. For example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used. Other,
Phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, etc. are used as necessary.

The culture temperature can be appropriately changed within the range in which the bacteria grow and produce pyruvate oxidase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C.
Although the culturing time varies depending on the conditions, it may be completed at a suitable time in consideration of the time when the maximum yield of pyruvate oxidase is reached, and it is usually about 12 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce pyruvate oxidase, but the pH is preferably about 6.0 to 8.0.

The pyruvate oxidase in the culture can be used as it is by collecting the culture medium containing the bacterial cells as it is. However, in general, when the pyruvate oxidase is present in the culture medium, the It is used after separating the pyruvate oxidase-containing solution and the microbial cells by filtration, centrifugation or the like. When pyruvate oxidase is present in the microbial cells, the obtained culture is collected by a means such as filtration or centrifugation, and then the microbial cells are collected by a mechanical method or an enzymatic method such as lysozyme. It is destroyed, and if necessary, a chelating agent such as EDTA and / or a surfactant is added to solubilize the pyruvate oxidase, and separated and collected as an aqueous solution.

The pyruvate oxidase-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the fractional precipitation method according to. The precipitate can then be dissolved in water and dialyzed through a semi-permeable membrane to remove lower molecular weight impurities. Further, the solution containing pyruvate oxidase, which is purified by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and obtained by using these means, is further purified by treatment such as vacuum concentration freeze-drying. To obtain the purified pyruvate oxidase.

The abbreviations for amino acids, peptides, nucleic acids, nucleic acid-related substances, etc. described in the present specification are based on their conventional abbreviations in the art, and examples thereof are listed below. In addition, all amino acids show L-form. DNA: Deoxyribonucleic acid RNA: Ribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Gln: Glutine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Glutamine: Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine

[0026]

EXAMPLES The present invention will be described in detail below with reference to examples.

[0027]

Example 1 [Separation of chromosomal DNA] Aerococcus viridans (IFO
The chromosomal DNA of 012219) was isolated by the following method, and the strain was shake-cultured in 150 ml of a normal broth medium (containing 0.5% sodium thiosulfate) at 37 ° C. overnight, followed by centrifugation (3,000).
(0 rotations for 10 minutes). 10% sucrose,
50 mM Tris-HCl (pH 8.0) 50 mM EDTA
Suspended in 5 ml of a solution containing 1 ml of lysozyme solution (10 mg / ml) and incubated at 37 ° C for 15 minutes, and then 1 ml of 10% SDS (sodium dodecyl sulfate) solution was added. Add an equal volume of chloroform-phenol mixture (1: 1) to the solution, stir and mix,
The aqueous layer and the solvent layer were separated by centrifugation at 30,000 rpm for 3 minutes, and the aqueous layers were combined.

The aqueous layer was gently overlaid with 2 volumes of ethanol, and the DNA was sprinkled on the glass rod while gently stirring with the glass rod to separate the DNA. Add 10 ml of 10 mM
It was dissolved in a solution containing Tris-HCl (pH 8.0) and 1 mM EDTA (hereinafter abbreviated as TE). After treating this with an equal volume of a chloroform-phenol mixed solution, the aqueous layer was separated by centrifugation, 2 volumes of ethanol was added, the DNA was separated again by the above method, and dissolved with 2 ml of TE.

[0029]

Example 2 [Separation of pACYC / 84 plasmid DNA] pACY
Escherichia coli pM191 possessing C / 84 (J.
Bacteriol 134 , 1141 (1981); ATCC3
7033) was shake-cultured in 1 L of BHI medium (manufactured by Difco). When the turbidity grew to OD ₆₆₀ = 1.0,
Spectinomycin (final concentration 300 μg / ml, when chloramphenicol was included as a plasmid resistance marker) was added and shaking was continued at 37 ° C. for 16 hours or more. The cells were collected by centrifugation at 3000 rpm for 10 minutes, and lysozyme-SDS method and cesium chloride-ethidium bromide method (Maniortis et al .: Molecular Clon).
Plasmid DNA was prepared according to ing pp 86-94 Cold Spring Harbor (1982).

[0030]

Example 3 [Preparation of plasmid pOXI3 having pyruvate oxidase (POP) gene] (i) Chromosomal DNA 2 of A. viridans prepared in Example 1
μl (about 0.5 μg) and 10 times concentration of EcoRI cleavage buffer (500 mM Tris-HCl (pH 7.5), 7
0 mM MgCl ₂ , 1 M NaCl, 70 mM mercaptoethanol 1 μl, EcoRI (Takara Shuzo 1.0 unit)
/ μl) 1 μl and water 6 μl were mixed and cut at 37 ° C. for 1 hour. About 0.3 μg of the separately prepared plasmid pACYC184 DNA was digested with EcoRI using the same method,
Further, 0.6 unit of alkaline phosphatase (hereinafter sometimes abbreviated as BAP; manufactured by Takara Shuzo) was added and treated at 65 ° C. for 1 hour. These two DNAs cut with EcoRI
The solution A was mixed, 1/10 amount of 3M sodium acetate was added, the mixture was further treated with a chloroform-phenol mixed solution in an amount equal to the total amount, and the aqueous layer was separated by centrifugation.

To this aqueous layer was added 2 volumes of ethanol, the DNA was precipitated by centrifugation and then dried under reduced pressure. 89 μl of water
After lysis with, a 10-fold concentration of ligation buffer (0.
5M Tris-HCl (pH 7.6), 0.1M MgCl ₂ ,
0.1 M dithiothreitol, 10 mM spermidine,
10 mM ATP) 10 μl and T ₄ DNA ligase 1 μl
(Takara Shuzo 175 unit) was added and mixed, and the mixture was left at 4 ° C. overnight. This DNA solution was subjected to chloroform-phenol treatment, ethanol precipitates were collected, dried under reduced pressure, and then dissolved with 10 μl of TE.

(Ii) 100 ml of BHI medium (Brain Hear
Escherichia coli W3110 strain in the logarithmic growth phase (stock number ME7778; ATCC, donated by the National Institute of Genetics) cultured in t Infusion, Difco)
27325] by centrifugation (10,000 r).
pm. 2 minutes) 40 ml ice-cold 30 mM potassium acetate, 100 mM RbCl, 10 mM CaCl ₂ , 50 m
A solution containing MMnCl ₂ and 15% glycerin (p
H5.8). After standing at 0 ° C. for 5 minutes, the mixture was centrifuged, the supernatant was removed, and further 4 ml of 10 mM MOPS buffer (manufactured by Dotite), 75 mM CaCl ₂ , 10 mM RbC was added.
1 and a solution containing 15% glycerin (pH 6.5)
And suspended at 0 ° C. for 15 minutes to prepare competent cells.

(Iii) Add 200 μl of this E. coli suspension to (i)
Add 10 μl of the DNA solution prepared in
Left. After adding 1 ml of BHI medium and incubating at 37 ° C for 90 minutes, 100 μl of this was added to tetracycline (15 μm).
g / ml) and spread on a BHI agar plate containing
Overnight to obtain a transformant. This transformant is
P medium (composition: 5 g of peptone, 2 g of meat extract, 5 g of yeast extract, 1 g of NaCl, 1 g of K ₂ HPO _4, 1 mg of MgS
O ₄ 0.5g, peroxidase 500IU, FAD
7.85 mg, dianisidine 0.1 g, thiamine pyrophosphate 424 mg, pyruvic acid 1 ml, agar 15
g, distilled water 1 liter, pH 7.0), and the plate was further replicated and cultured at 37 ° C. overnight.

When the transformation of about 4500 colonies was examined, one strain was obtained in which the periphery of the colonies was discolored to dark brown, and this strain was designated Escherichia coli W3110.pOXI.
3 strains "Microorganism Accession No.
RMP-No. 9071, Jiken Kenjo No. 1565, FER
MBP-1565 ". This bacterium was purely isolated and then cultured overnight in a BHI medium at 37 ° C., and when the productivity of pyruvate oxidase was examined by the pyruvate oxidase activity measurement method described later, it had a pyruvate oxidase activity of about 3 u / ml. . The plasmid possessed by this strain was isolated in the same manner as in Example 2,
Containing the pyruvate oxidase gene, pACYC18
The plasmid containing the four genes was named pOXI3.

Method for Measuring Pyruvate Oxidase Activity The method for measuring the activity of pyruvate oxidase of the present invention is as follows. 0.5 M potassium pyruvate 0.1 ml 0.5 M phosphate buffer (pH 7.0) 0.2 ml 0.2% 4-aminoantipyrine 0.1 ml 0.2% N, N-dimethylaniline 0.2 ml 10 mM MgCl ₂ 50 μl 10 mM thiaminopyrophosphate 20 μl peroxidase (45 U / ml) 0.1 ml 1 mM FAD 10 μl distilled water 0.22 ml 1.0 ml of the reaction solution having the above composition was collected in a test tube, and 37
After preheating at 3 ° C for 3 minutes, 20 µl of enzyme solution was added and
The reaction was carried out at 7 ° C. for 10 minutes.
The reaction was stopped by adding 1M EDTA (pH 7.5), and then 1.7 ml of distilled water was added thereto to give a purple color of 5
The colorimetry is performed at a wavelength of 65 nm. The activity to produce 1 μmole of hydrogen peroxide per minute was defined as 1 unit (U).

[0036]

Example 4 [Mapping of pOXI3 and Determination of Nucleotide Sequence of POP Gene] pOXI3 plasmid DNA was prepared from Escherichia coli W3110 pOXI3 strain by the same method as pACYC184. Restriction enzymes ClaI, EcoRV, HindIII, Sca for pOXI3DNA
A cut map with I, PstI, PvuII, and XbaI (all manufactured by Takara Shuzo) and Hpal (manufactured by Toyobo) was prepared.
The result is shown in FIG. The nucleotide sequence of DNA containing the pyruvate oxidase gene was determined by the dideoxy method using M13 phage (Science 214 1205-1210).
(1981)). The nucleotide sequence and amino acid sequence of the POP structural gene are shown in FIG. 2 and FIG.
Is a continuation of FIG. 2).

[0037]

Example 5 [Production of pyruvate oxidase] Escherichia coli W3110pOXI3 strain was mixed with 20 l of BHI medium (Dif.
Co.) at 37 ° C. for 18 hours with a jar fermeter, and the cells were collected by centrifugation at 5,000 rpm for 10 minutes. After washing with 2 l of physiological saline, it was suspended with 2 l of 10 mM phosphate buffer (pH 7.0). 1mg lysozyme
/ Ml, 2 mM EDTA-1Na, Triton X-10
0 was added to 0.1% and kept at 37 ° C. for 30 minutes, and the supernatant was separated by centrifugation at 5,000 rpm for 10 minutes. 1.9 l of the supernatant was salted out with ammonium sulfate (40% to 6%).
6%), and the precipitate is centrifuged (5,000 rpm 30
Min). The precipitate is washed with 200 ml of 10 mM
It was dissolved in a phosphate buffer (pH 7.0, containing 10 μM FAD), and desalted with Sephadex G-25. After that, ion exchange chromatography was performed using DEAE-Sepharose CL-6B to fractionate the active fraction, followed by desalting.
A powder preparation was obtained by freeze-drying. This enzyme preparation 51u measured by the pyruvate oxidase activity measuring method described above.
/ Mg specific activity.

[0038]

[Example 6] [Determination of amino acid sequence of amino-terminal portion of pyruvate oxidase separated and purified from recombinant] Regarding pyruvate oxidase separated and purified in Example 5, an amino acid sequencer (Beckman System 980M manufactured by Beckman) was used.
The sequence of 10 amino acids from the amino terminus was examined using E).

As a result, Met was present at the N-terminus in about 80% of the products, and SerAsn Lys Ile Asn Ile from the 1st position of the amino acid sequence.
It was confirmed that the sequence was Gly Leu (Ala).

[0040]

INDUSTRIAL APPLICABILITY The present invention clarified the pyruvate oxidase gene and the amino acid sequence of pyruvate oxidase, and provided an efficient method for producing pyruvate oxidase by a genetic engineering technique. Further, by using the pyruvate oxidase gene of the present invention and various genetic engineering techniques, a more efficient method for producing pyruvate oxidase can be brought about.

[0041]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 591 amino acid residues Sequence type: Peptide Sequence name: Pyruvate oxidase amino acid sequence Sequence Ser Asp Asn Lys Ile Asn Ile Gly Leu Ala Val Met Lys Ile Leu 15 Glu Ser Trp Gly Ala Asp Thr Ile Tyr Gly Ile Pro Ser Gly Thr 30 Leu Ser Ser Leu Met Asp Ala Met Gly Glu Glu Glu Asn Asn Val 45 Lys Phe Leu Gln Val Lys His Glu Glu Val Gly Ala Met Ala Ala 60 Val Met Gln Ser Lys Phe Gly Gly Asn Leu Gly Val Thr Val Gly 75 Ser Gly Gly Pro Gly Ala Ser His Leu Ile Asn Gly Leu Tyr Asp 90 Ala Ala Met Asp Asn Ile Pro Val Val Ala Ile Leu Gly Ser Arg 105 Pro Gln Arg Glu Leu Asn Met Asp Ala Phe Gln Glu Leu Asn Gln 120 Asn Pro Met Tyr Asp His Ile Ala Val Tyr Asn Arg Arg Val Ala 135 Tyr Ala Glu Gln Leu Pro Lys Leu Val Asp Glu Ala Ala Arg Met 150 Ala Ile Ala Lys Arg Gly Val Ala Val Leu Glu Val Pro Gly Asp 165 Phe Ala Lys Val Glu Ile Asp Asn Asp Gln Trp Tyr Ser Ser Ala 180 Asn Ser Leu Arg Lys Tyr Ala Pro Ile Ala Pro Ala Ala Gln Asp 19 5 Ile Asp Ala Ala Val Glu Leu Leu Asn Asn Ser Lys Arg Pro Val 210 Ile Tyr Ala Gly Ile Gly Thr Met Gly His Gly Pro Ala Val Gln 225 Glu Leu Ala Arg Lys Ile Lys Ala Pro Val Ile Thr Thr Gly Lys 240 Asn Phe Glu Thr Phe Glu Trp Asp Phe Glu Ala Leu Thr Gly Ser 255 Thr Tyr Arg Val Gly Trp Lys Pro Ala Asn Glu Thr Ile Leu Glu 270 Ala Asp Thr Val Leu Phe Ala Gly Ser Asn Phe Pro Phe Ser Glu 285 Val Glu Gly Thr Phe Arg Asn Val Asp Asn Phe Ile Gln Ile Asp 300 Ile Asp Pro Ala Met Leu Gly Lys Arg His His Ala Asp Val Ala 315 Ile Leu Gly Asp Ala Gly Leu Ala Ile Asp Glu Ile Leu Asn Lys 330 Val Asp Ala Val Glu Glu Ser Ala Trp Trp Thr Ala Asn Leu Lys 345 Asn Ile Ala Asn Trp Arg Glu Tyr Ile Asn Met Leu Glu Thr Lys 360 Glu Glu Gly Asp Leu Gln Phe Tyr Gln Val Tyr Asn Ala Ile Asn 375 Asn His Ala Asp Glu Asp Ala Ile Tyr Ser Ile Asp Val Gly Asn 390 Ser Thr Gln Thr Ser Ile Arg His Leu His Met Thr Pro Lys Asn 405 Met Trp Arg Thr Ser Pro Leu Phe Ala Thr Met Gly Ile Ala Ile 420 Pro Gly Gly Leu Gly Ala Lys Asn Thr Ty r Pro Asp Arg Gln Val 435 Trp Asn Ile Ile Gly Asp Gly Ala Phe Ser Met Thr Tyr Pro Asp 450 Val Val Thr Asn Val Arg Tyr Asn Met Pro Val Ile Asn Val Val 465 Phe Ser Asn Thr Glu Tyr Ala Phe Ile Lys Asn Lys Tyr Glu Asp 480 Thr Asn Lys Asn Leu Phe Gly Val Asp Phe Thr Asp Val Asp Tyr 495 Ala Lys Ile Ala Glu Ala Gln Gly Ala Lys Gly Phe Thr Val Ser 510 Arg Ile Glu Asp Met Asp Arg Val Met Ala Glu Ala Val Ala Ala 525 Asn Lys Ala Gly His Thr Val Val Ile Asp Cys Lys Ile Thr Gln 540 Asp Arg Pro Ile Pro Val Glu Thr Leu Lys Leu Asp Ser Lys Leu 555 Tyr Ser Glu Asp Glu Ile Lys Ala Tyr Lys Glu Arg Tyr Glu Ala 570 Ala Asn Leu Val Pro Phe Arg Glu Tyr Leu Glu Ala Glu Gly Leu 585 Glu Ser Lys Tyr Ile Lys 591

[0042]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 1773 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin of organism Name: Aerococcus viridans Strain name: IFO012219 Sequence characteristics : Pyruvate oxidase gene sequence TCA GAT AAC AAA ATT AAC ATC GGT TTA GCA GTT ATG AAG ATT TTA 45 GAA TCT TGG GGA GCA GAT ACT ATT TAT GGT ATT CCT TCA GGT ACT 90 TTA AGC TCA TTA ATG GAT GCT ATG GGT GAA GAA GAA AAC AAC GTC 135 AAA TTC CTA CAA GTG AAA CAC GAA GAA GTA GGT GCA ATG GCT GCT 180 GTA ATG CAA AGC AAA TTC GGC GGT AAC TTA GGT GTT ACT GTA GGT 225 TCT GGT GGA CCA GGT GCA TCT CAC TTG ATC AAC GGT TTA TAC GAT 270 GCA GCA ATG GAT AAC ATT CCA GTA GTT GCG ATC TTA GGT TCT CGT 315 CCA CAA CGC GAA TTA AAC ATG GAC GCT TTC CAA GAA TTA AAC CAG 360 AAC CCA ATG TAC GAC CAT ATT GCA GTT TAC AAC CGT CGT GTA GCT TAT GCT GAG CAA TTA CCA AAA TTA GTT GAC GAA GCA GCT CGT ATG 450 GCT ATC GCT AAA C GC GGT GTA GCA GTT CTA GAA GTA CCT GGT GAT 495 TTT GCT AAA GTT GAA ATC GAC AAC GAC CAA TGG TAT TCA TCT GCA 540 AAC AGC TTA CGT AAA TAT GCA CCA ATC GCT CCA GCA GCA CAA GAT 585 ATC GAC GCA GCA GTT GAA TTA TTA AAC AAC TCT AAA CGT CCA GTT 630 ATC TAC GCT GGT ATT GGT ACT ATG GGC CAC GGT CCT GCA GTT CAA 675 GAA TTA GCT CGT AAA ATC AAA GCG CCA GTT ATC ACT ACT GGT AAA 720 AAC TTT GAA ACT TTC GAG TGG GAT TTC GAA GCG TTA ACA GGT TCT 765 ACT TAT CGT GTA GGT TGG AAA CCA GCT AAC GAA ACA ATT TTA GAA 810 GCT GAC ACA GTA TTA TTT GCT GGT TCA AAC TTC CCA TTC TCA GAG 855 GTT GAA GGT ACT TTC CGT AAC GTG GAT GAT GAC TTC ATC CAA ATC GAT 900 ATC GAC CCA GCT ATG TTA GGT AAA CGT CAC CAC GCT GAT GTT GCT 945 ATC TTA GGT GAT GCT GGT CTA GCA ATC GAC GAA ATC TTA AAC AAA 990 GTA GAT GCT GTT GAA GAG TCA GCA TGG TGG ACA GCT AAC TTG AAA 1035 AAC ATC GCT AAC TGG CGT GAA TAC ATC AAC ATG TTA GAA ACT AAA 1080 GAA GAA GGC GAC TTG CAA TTC TAC CAA GTG TAC AAT GCA ATC AAC 1125 AAC CAC GCC GAC GAA GAT GCA ATC TAC TCT ATT GAT GTT GCA GT AAC 1170 TCA ACT CAA ACT TCT ATC CGT CAT TTA CAT ATG ACA CCT AAA AAC 1215 ATG TGG AGA ACT TCT CCA TTA TTC GCG ACA ATG GGT ATC GCT ATC 1260 CCT GGT GGT TTA GGT GCT AAA AAC ACT TAC CCA GAT CGT CAA GTT 1305 TGG AAC ATC ATC GGT GAT GGT GCT TTC TCT ATG ACT TAC CCA GAT 1350 GTA GTA ACT AAC GTA CGT TAC AAC ATG CCT GTA ATC AAC GTT GTA 1395 TTC TCT AAC ACT GAA TAT GCC TTC ATC AAA AAC AAA TAT GAA GAC 1440 AAC AAA AAC TTA TTC GGT GTA GAC TTT ACA GAT GTT GAT TAC 1485 GCT AAA ATC GCT GAA GCA CAA GGT GCT AAA GGA TTT ACT GTA AGC 1530 CGT ATC GAA GAT ATG GAC CGT GTA ATG GCT GAA GCT GTT GCA GCT 1575 AAT AAA GCA GGT CAC ACT GTC GTT ATC GAC TGT AAG ATT ACT CAA 1620 GAT CGT CCA ATC CCT GTA GAA ACA TTG AAA TTA GAT TCA AAA CTT 1665 TAC AGC GAA GAC GAA ATC AAA GCT TAC AAA GAA CGC TAC GAA GCT 1710 GCT AAC TTA GTA CCA TTC AGA GAG TAC TTA GAA GCT GAA GGC TTA 1755 GAA TCT AAA TAC ATC AAA 1773

[Brief description of the drawings]

FIG. 1 shows a schematic diagram showing the construction of the pOXI3 vector.

FIG. 2 is a pyruvate oxidase gene DNA.
The respective sequences of the coding strand (5 ′ → 3 ′) and the resulting translation product are shown.

FIG. 3 shows the respective sequences of the coding strand of pyruvate oxidase gene DNA (5 ′ → 3 ′) and the resulting translation product following FIG. 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9 / 02 C12R 1:19)

Claims (4)

[Claims] 1. A polydeoxyribonucleic acid that is a pyruvate oxidase gene encoding the following amino acid sequence from the N-terminal side, or a recombinant DNA in which an expression vector contains polydeoxyribonucleic acid that is a constituent component of the gene.
To a host microorganism to obtain a transformant, the transformant is cultured to express the genetic information of the polydeoxyribonucleic acid, and then the polypeptide which is pyruvate oxidase or the pyruvate oxidase is a constituent component. A method for producing pyruvate oxidase, which comprises collecting a polypeptide. A-Ser Asp Asn Lys Ile Asn Ile Gly Leu Ala Val Met Lys Ile Leu Glu Ser Trp Gly Ala Asp Thr Ile Tyr Gly Ile Pro Ser Gly Thr Leu Ser Ser Leu Met Asp Ala Met Gly Glu Glu Glu Aslu Asn Val Lys Phe Leu Gln Val Lys His Glu Glu Val Gly Ala Met Ala Ala Val Met Gln Ser Lys Phe Gly Gly Asn Leu Gly Val Thr Val Gly Ser Gly Gly Pro Gly Ala Ser His Leu Ile Asn Gly Leu Tyr Asp Ala Ala Met Asp Asn Ile Pro Val Val Ala Ile Leu Gly Ser Arg Pro Gln Arg Glu Leu Asn Met Asp Ala Phe Gln Glu Leu Asn Gln Asn Pro Met Tyr Asp His Ile Ala Val Tyr Asn Arg Arg Val Ala Tyr Ala Glu Gln Leu Pro Lys Leu Val Asp Glu Ala Ala Arg Met Ala Ile Ala Lys Arg Gly Val Ala Val Leu Glu Val Pro Gly Asp Phe Ala Lys Val Glu Ile Asp Asn Asp Gln Trp Tyr Ser Ser Ala Asn Ser Leu Arg Lys Tyr Ala Pro Ile Ala Pro Ala Ala Gln Asp Ile Asp Ala Ala Val Glu Leu Leu Asn Asn Ser Lys Arg Pro Val Ile Tyr Ala Gly Ile Gly Thr Met Gly His Gly Pro Ala Val Gln Glu Leu Ala Arg Lys Ile Lys Ala Pro Val Ile Thr Thr Gly Lys Asn Phe Glu Thr Phe Glu Trp Asp Ph e Glu Ala Leu Thr Gly Ser Thr Tyr Arg Val Gly Trp Lys Pro Ala Asn Glu Thr Ile Leu Glu Ala Asp Thr Val Leu Phe Ala Gly Ser Asn Phe Pro Phe Ser Glu Val Glu Gly Thr Phe Arg Asn Val Asp Asn Phe Ile Gln Ile Asp Ile Asp Pro Ala Met Leu Gly Lys Arg His His Ala Asp Val Ala Ile Leu Gly Asp Ala Gly Leu Ala Ile Asp Glu Ile Leu Asn Lys Val Asp Ala Val Glu Glu Ser Ala Trp Trp Thr Ala Asn Leu Lys Asn Ile Ala Asn Trp Arg Glu Tyr Ile Asn Met Leu Glu Thr Lys Glu Glu Gly Asp Leu Gln Phe Tyr Gln Val Tyr Asn Ala Ile Asn Asn His Ala Asp Glu Asp Ala Ile Tyr Ser Ile Asp Val Gly Asn Ser Thr Gln Thr Ser Ile Arg His Leu His Met Thr Pro Lys Asn Met Trp Arg Thr Ser Pro Leu Phe Ala Thr Met Gly Ile Ala Ile Pro Gly Gly Leu Gly Ala Lys Asn Thr Tyr Pro Asp Arg Gln Val Trp Asn Ile Ile Gly Asp Gly Ala Phe Ser Met Thr Tyr Pro Asp Val Val Thr Asn Val Arg Tyr Asn Met Pro Val Ile Asn Val Val Phe Ser Asn Thr Glu Tyr Ala Phe Ile Lys Asn Lys Tyr Glu Asp Thr Asn Lys Asn Leu Phe Gly Val Asp Phe Thr Asp Val Asp Tyr Ala Lys Ile Al a Glu Ala Gln Gly Ala Lys Gly Phe Thr Val Ser Serg Ile Glu Asp Met Asp Arg Val Met Ala Glu Ala Val Ala Ala Asn Lys Ala Gly His Thr Val Val Ile Asp Cys Lys Ile Thr Gln Asp Arg Pro Ile Pro Val Glu Thr Leu Lys Leu Asp Ser Lys Leu Tyr Ser Glu Asp Glu Ile Lys Ala Tyr Lys Glu Arg Tyr Glu Ala Ala Asn Leu Val Pro Phe Arg Glu Tyr Leu Glu Ala Glu Gly Leu Glu Ser Lys Tyr Ile Lys -B amino acid residues, represents a hydrogen atom or an acetyl group, B denotes an amino acid residue, -OH or -NH _2]. 2. The method according to claim 1, wherein the polydeoxyribonucleic acid comprises a pyruvate oxidase gene derived from a microorganism belonging to the Lactobacillusaceae family or the Streptococcus family, or the gene as a constituent component. 3. The method according to claim 2, wherein the pyruvate oxidase gene derived from a microorganism belonging to the Streptococcus family is a polydeoxyribonucleic acid obtained from the pyruvate oxidase-producing bacterium of the genus Aerococcus. 4. The polydeoxyribonucleic acid has the formula X-TCA GAT AAC AAA ATT AAC ATC GGT TTA GCA GTT ATG AAG ATT TTA GAA TCT TGG GGA GCA GAT ACT ATT TAT GGT ATT CCT TCA GGT ACT TTA AGC. TCA TTA ATG GAT GCT ATG GGT GAA GAA GAA AAC AAC GTC AAA TTC CTA CAA GTG AAA CAC GAA GAA GTA GGT GCA ATG GCT GCT GTA ATG CAA AGC AAA TTC GGC GGT AAC TTA GGT GTT ACT GTA GGT TCT GGT GGA CCA GGT GCA TCT CAC TTG ATC AAC GGT TTA TAC GAT GCA GCA ATG GAT AAC ATT CCA GTA GTT GCG ATC TTA GGT TCT CGT CCA CAA CGC GAA TTA AAC ATG GAC GCT TTC CAA GAA TTA AAC CAG AAC CCA ATG TAC GAC CAT ATT GCA GTT TAC ATAC CGT GTA GCT TAT GCT GAG CAA TTA CCA AAA TTA GTT GAC GAA GCA GCT CGT ATG GCT ATC GCT AAA CGC GGT GTA GCA GTT CTA GAA GTA CCT GGT GAT TTT GCT AAA GTT GAA ATC GAC AAC GAC CAA TGG TAT TCA TCT GCA AAC AGC TTA CGT AAA TAT GCA CCA ATC GCT CCA GCA GCA CAA GAT ATC GAC GCA GCA GTT GAA TTA TTA AAC AAC TCT AAA CGT CCA GTT ATC TAC GCT GGT ATT GGT ACT ATG GGC CAC GGT CCT GCA GTT CAA GAA TTA GCT CGT AAA ATC AAA GCG CCA GTT ATC ACT ACT GGT AAA AAC TTT GAA ACT TTC GAG TGG GAT TTC GAA GCG TTA ACA GGT TCT ACT TAT CGT GTA GGT TGG AAA CCA GCT AAC GAA ACA ATT TTA GAA GCT GAC ACA GTA TTA TTT GCT GGT TCA AAC TTC CCA TTC TCA GAG GTT GAA GGT ACT TTC CGT AAC GTG GAT AAC TTC ATC CAA ATC GAT ATC GAC CCA GCT ATG TTA GGT AAA CGT CAC CAC GCT GAT GTT GCT ATC TTA GGT GAT GCT GGT CTA GCA ATC GAC GAA ATC TTA AAA GTA GAT GCT GTT GAA GAG TCA GCA TGG TGG ACA GCT AAC TTG AAA AAC ATC GCT AAC TGG CGT GAA TAC ATC AAC ATG TTA GAA ACT AAA GAA GAA GGC GAC TTG CAA TTC TAC CAA GTG TAC AAT GCA ATC AAC AAC CAC GCC GAC GAC GAA GAT GCA ATC TAC TCT ATT GAT GTT GGT AAC TCA ACT CAA ACT TCT ATC CGT CAT TTA CAT ATG ACA CCT AAA AAC ATG TGG AGA ACT TCT CCA TTA TTC GCG ACA ATG GGT ATC GCT ATC CCT GGT GGT TTA GGT GCT AAA AAC TAC CCA GAT CGT CAA GTT TGG AAC ATC ATC GGT GAT GGT GCT TTC TCT ATG ACT TAC CCA GAT GTA GTA ACT AAC GTA CGT TAC AAC ATG CCT GTA ATC AAC GTT GTA TTC TCT AAC ACT GAA TAT GCC TTC ATC AAA AAC AAA TAT GAA GAC ACT AAC AAA AAC TTA TTC GGT GTA GAC TTT ACA GAT GTT GAT TAC GCT AAA ATC GCT GAA GCA CAA GGT GCT AAA GGA TTT ACT GTA AGC CGT ATC GAA GAT ATG GAC CGT GTA ATG GCT GAA GCT GTT GCA GCT AAT AAA GCA GGT CAC ACT GTC GTT ATC GAC TGT AAG ATT ACT CAA GAT CGT CCA ATC CCT GTA GAA ACA TTG AAA TTA GAT TCA AAA CTT TAC AGC GAA GAC GAA ATC AAA GCT TAC AAA GAA CGC TAC GAA GCT GCT AAC TTA GTA CCA TTC AGA GAG TAC 4. TTA GAA GCT GAA GGC TTA GAA TCT AAA TAC ATC AAA-Y [wherein X represents a codon or hydrogen atom other than TAA, TAG and TGA, and Y represents a codon or hydrogen atom]. Manufacturing method.