JPWO2007066430A1 - Peptide production method - Google Patents

Abstract

According to the present invention, a protein having peptide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, the transformant, etc. There are provided a method for producing a protein having peptide synthesis activity, a method for producing a peptide using the protein, and a method for producing a peptide using a transformant or a microorganism culture producing the protein as an enzyme source. The

Description

  The present invention relates to a protein having peptide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, a method for producing the protein, and the protein The present invention relates to a method for producing a peptide using the above-described method, and a method for producing a peptide using a microorganism or a transformant that produces the protein.

As for peptide mass synthesis methods, chemical synthesis methods (liquid phase method, solid phase method), enzymatic synthesis methods, and biological synthesis methods using DNA recombination methods are known. Currently, enzymatic synthesis or biological synthesis is mainly used for peptides with long chains of several tens of residues or more, and chemical synthesis and enzymatic synthesis for peptides with short chains of 2 to several residues. Is mainly used.
The synthesis of short-chain peptides by chemical synthesis requires operations such as functional group protection and deprotection, and racemates are also produced as by-products, so chemical synthesis is an economical and efficient method. I can't say that.

  For the synthesis of short-chain peptides by enzymatic methods, methods utilizing the reverse reaction of proteolytic enzymes (proteases) (see Non-Patent Document 1) and transesterification enzymes (Patent Documents 1 to 3, Non-Patent Document 2) are used. A method using a thermostable aminoacyl-t-RNA synthetase (Patent Documents 4 to 7), a method using a non-ribosomal peptide synthetase (hereinafter referred to as NRPS) (Non-Patent Documents 3 and 4 and Patent Document 8) 9) is known.

  However, the method using the reverse reaction of proteolytic enzyme requires the protection and deprotection of the functional group of the amino acid serving as the substrate, and there is a problem that it is difficult to increase the efficiency of the peptide formation reaction and to prevent the peptide decomposition reaction. . In the method using transesterification enzyme, esterification of an amino acid serving as a substrate is necessary, and there are problems such as improvement in efficiency and a decrease in yield due to decomposition of the amino acid ester serving as a substrate and the produced peptide. The method using a thermostable aminoacyl t-RNA synthetase has a problem that it is difficult to prevent enzyme expression and by-product reactions other than the target product. Regarding the method using NRPS, it is difficult to express the enzyme using a DNA recombination method because the enzyme molecule is huge, and the coenzyme 4′-phosphopantetheine (4′-phosphopantetheine) is used. ) Is necessary and is not an efficient manufacturing method.

  On the other hand, γ-glutamylcysteine synthetase, D-alanyl-D-alanine (D-Ala-D-Ala), whose enzyme molecular weight is smaller than NRPS and does not require coenzyme 4'-phosphopantetheine A group of peptide synthetases such as ligase (D-Ala-D-Ala ligase) and poly-γ-glutamate synthetase are also known. Most of these enzymes use D-amino acid as a substrate or catalyze the formation of peptide bond at the γ-position carboxyl group, so the short chain that binds peptide at the α-position carboxyl group of L-amino acid. It cannot be used for peptide synthesis.

  Only basilicin synthase and glutathione synthetase are known to have peptide bond-forming activity at the α-carboxyl group of L-amino acids. Basilisine synthases derived from microorganisms belonging to the genus Bacillus are dipeptide antibiotics bacillicin [L-Ala-L-anticapsin] and L-alanyl-L-alanine (L-Ala-L). -Ala) has been reported to have an activity to synthesize various dipeptides from the same or different free amino acids in various combinations (see Patents 5 and 6). Document 10, Non-Patent Document 7). Glutathione synthetase is a glutathione (γ-L-Glu-L) by linking cysteine residue of γ-L-glutamyl-L-cysteine (γ-L-Glu-L-Cys) and glycine (Gly) at the α-position Γ-L-glutamyl dipeptide (tripeptide) by linking glycine to the C-terminal amino acid residues of several γ-L-glutamylamino acids (dipeptides) Non-patent document 8) has not reported an activity of linking γ-L-glutamylamino acid to L-amino acids other than glycine.

It is possible to produce various dipeptides and tripeptides using these enzymes, but due to the substrate specificity of these enzymes, there are some short-chain peptides that are not sufficiently efficient to produce these enzymes. There is a need for new short-chain peptide synthases with different substrate specificities.
The nucleotide sequence of the chromosomal DNA of Clostridium acetobutylicum ATCC 824 and the deduced nucleotide sequence of the gene are also known (see Non-Patent Document 9). However, as well as the function of the protein encoded by the CAC1540 gene in the gene, it is not known whether the CAC1540 gene is a gene encoding a protein having an actual function.
International Publication No. 2003/010187 Pamphlet International Publication No. 2003/010307 Pamphlet International Publication No. 2003/010189 Pamphlet JP 58-146539 A JP 58-209991 A JP 58-209992 JP JP 59-106298 A U.S. Patent No. 5795738 U.S. Patent No. 5562116 International Publication No. 2004/058960 Pamphlet J. Biol. Chem., 119, 707-720 (1937) J. Biotechnol., 115, 211-220 (2005) Chem. Biol., 7, 373-384 (2000) FEBS Lett., 498, 42-45 (2001) J. Ind. Microbiol., 2, 201-208 (1987) Enzyme Microb. Technol., 29, 400-406 (2001) J. Bacteriol., 187, 5195-5202 (2005) J. Biol. Chem., 254, 5184-5190 (1979) http://gib.genes.nig.ac.jp/single/index.php?spid=Cace_ATCC824

  An object of the present invention is to provide a protein having peptide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, the transformant, etc. An object of the present invention is to provide a method for producing the protein used, a method for producing a peptide using the protein, and a method for producing a peptide using a transformant or a microorganism culture producing the protein as an enzyme source.

The present invention relates to the following (1) to (14).
(1) The protein according to any one of [1] to [3] below.
[1] A protein having the amino acid sequence represented by SEQ ID NO: 1 [2] The amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and peptide synthesis activity [3] a protein having the amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having peptide synthesis activity (2) Any of [1] to [3] below The DNA according to the above.
[1] DNA encoding the protein of (1) above
[2] DNA having the base sequence represented by SEQ ID NO: 2
[3] DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having peptide synthesis activity
(3) A recombinant DNA containing the DNA of (2) above.
(4) A transformant having the recombinant DNA of (3) above.
(5) The transformant according to (4) above, wherein the transformant is a transformant obtained using a microorganism as a host.
(6) The transformant according to (5) above, wherein the microorganism belongs to the genus Escherichia .
(7) A microorganism having the ability to produce the protein of (1) above is cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture. Manufacturing method.
(8) The method for producing the protein of (7) above, wherein the microorganism having the ability to produce the protein of (1) is any one of the transformants of (4) to (6) above.
(9) A culture of a microorganism having the ability to produce the protein of (1) above, a processed product of the culture, or the protein of (1) above and one or more amino acids or amino acids and dipeptides in an aqueous medium A method for producing a peptide, wherein the peptide is produced, accumulated in the medium, and collected from the medium.
(10) The method for producing a peptide of (9) above, wherein the amino acid is an amino acid selected from the group consisting of L-amino acid, glycine, and β-alanine.
(11) L-amino acid is L-alanine, L-glutamine, L-glutamic acid, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine , L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, L-6-diazo-5-oxo-norleucine, N-acetyl-L-cysteine and N-acetyl-L-α-amino The method for producing a peptide according to the above (10), which is an L-amino acid selected from the group consisting of butyric acid.
(12) The dipeptide is γ-L-glutamyl amino acid, γ-D-glutamyl amino acid, γ-L- (α-methyl) glutamyl amino acid, β-aminoglutaryl amino acid, γ-L- (N-methyl) glutamyl amino acid, The method for producing a peptide according to the above (9), which is a dipeptide selected from the group consisting of γ-DL- (β-methyl) glutamylamino acid and γ-L- (γ-methyl) glutamylamino acid.
(13) γ-L-glutamyl amino acid is γ-L-glutamyl-L-cysteine, γ-L-glutamyl-L-α-aminobutyric acid, γ-L-glutamyl-LS-methylcysteine, γ-L-glutamyl -L-serine, γ-L-glutamyl-L-alanine, γ-L-glutamyl-L-norvaline, γ-L-glutamyl-L-hypoglycine, γ-L-glutamyl-β-cyanoalanine, γ-L The method for producing a peptide according to (12), which is a γ-L-glutamyl amino acid selected from the group consisting of -glutamyl-Se-methylselenocysteine and N- (γ-L-glutamyl) amino-D-proline.
(14) Any of (9) to (13) above, wherein the microorganism having the ability to produce the protein of (1) is the transformant according to any one of (4) to (6) above The manufacturing method of the peptide as described in one.

  According to the present invention, a protein having peptide synthesis activity can be produced, and various peptides can be produced using the protein or a transformant or a microorganism having an ability to produce the protein.

FIG. 3 is a diagram showing the construction process of plasmid pCAC1540.

Explanation of symbols

CAC1540 in the figure, CAC1540 gene derived from Clostridium acetobutylicum ATCC824 strain, P T7 represents a T7 promoter gene, His-tag is His-tag sequence.

1. 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 peptide synthesis activity, and [3] represented by SEQ ID NO: 1 A protein having an amino acid sequence having 80% or more homology with the amino acid sequence and having peptide synthesis activity can be mentioned.

The peptide synthesis activity in the present specification is an activity to form a peptide bond, an activity to form a peptide bond between the carboxyl group at the α-position of an amino acid and the amino group of another amino acid, and an amino acid at the C-terminal of a dipeptide The activity of forming a peptide bond between the carboxyl group at the α-position of the residue and the amino group of the amino acid.
In the amino acid sequence represented by SEQ ID NO: 1, a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added and having peptide synthesis activity is Molecular cloning, a laboratory manual, Third Edition, Cold Spring. Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Cloning 3rd 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), Using site-directed mutagenesis described in Proc. Natl. Acad. Sci. USA, 82 , 488 (1985), etc., for example, site specific to DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. By introducing genetic mutations 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, Preferably it is 1-20, More preferably, it is 1-10, More preferably, it is 1-5.
In the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids are deleted, substituted or added, even if one or more amino acids are deleted, substituted or added at any position in the same sequence. Good.

  Deletions, substitutions or additions may occur simultaneously, and the amino acids substituted or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Examples of amino acids that can be substituted with each other are shown below. 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 Group 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: phenylalanine, tyrosine In order for the protein of the present invention to have peptide synthesis activity, does it have the amino acid sequence represented by SEQ ID NO: 1? Or the amino acid sequence represented by SEQ ID NO: 1 Comparison 80% in the, preferably 90% or more, more preferably 94% or more, more preferably 98% or more, particularly preferably desirable to have less than 99% homology.

The homology of amino acid sequences and base sequences is based on the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods Enzymol., 183 , 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

As a means for confirming that the protein of the present invention is a protein having peptide synthesis activity, for example, a transformant that expresses the protein of the present invention using a DNA recombination method is prepared, and the transformant is used. After producing the protein of the present invention, the protein of the present invention and one or more amino acids, or dipeptide and amino acid, preferably γ-glutamyl amino acid and L-amino acid, glycine, and β-alanine And ATP can be present in an aqueous medium, and whether or not a peptide is produced and accumulated in the aqueous medium can be analyzed and confirmed by HPLC or the like.
2. 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 or [3] DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2; DNA encoding a protein having peptide synthesis activity,
Can give.

  The term “hybridize” as used herein refers to a step in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the base sequence is useful as a probe for Northern or Southern blot analysis, but the probe used for hybridization can be used as an oligonucleotide primer for PCR analysis. It may be DNA. Examples of DNA used as a probe include DNAs of at least 10 bases, preferably 15 bases or more, more preferably 100 bases or more, still more preferably 200 bases or more, and particularly preferably 500 bases or more.

  Methods for DNA hybridization experiments are well known and described in, for example, Molecular Cloning 3rd Edition (2001), Methods for General and Molecular Bacteriolgy, ASM Press (1994), Immunology methods manual, Academic press (Molecular). In addition, hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

  The above stringent conditions include, for example, a DNA-immobilized filter and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ), Incubated in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA overnight at 42 ° C., for example, in a 0.2 × SSC solution at about 65 ° C. The conditions for washing the filter can be raised. Stringent conditions can be adjusted according to the length of the chain length of the probe DNA and the GC content, and can be set by the method described in Molecular Cloning 3rd edition and the like. Lower stringent conditions can also be used, but stringent conditions can be changed by adjusting the formamide concentration (lower stringent concentration lowers stringency), changing salt concentration and temperature conditions. is there. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / L sodium chloride, 0.2 mol / L sodium dihydrogen phosphate, 0.02 mol / L EDTA, pH 7.4), 0.5% Incubate overnight at 37 ° C in a solution containing SDS, 30% formamide, 100 μg / L denatured salmon sperm DNA, and then wash with 50 ° C 1x SSC, 0.1% SDS solution. I can give you. Further, examples of lower stringent conditions include conditions in which hybridization is performed under the above-described low stringency conditions and then washed with a solution having a high salt concentration (for example, 5 × SSC).

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. 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, the DNA of [1] or [2] described above when calculated based on the above parameters using a program such as BLAST and FASTA described above. DNA having at least 80% or more, preferably 90% or more, more preferably 94% or more, still more preferably 98% or more, and particularly preferably 99% or more can be mentioned.

The fact that the DNA that hybridizes with the above-described DNA under stringent conditions is a DNA that encodes a protein having peptide synthesis activity. This means that a recombinant DNA that expresses the DNA is prepared, The protein is purified from a culture obtained by culturing a microorganism obtained by introduction into a host cell, and the purified protein is used as an enzyme source. The enzyme source and one or more amino acids, or a dipeptide and an amino acid, preferably Is an amino acid selected from the group consisting of γ-glutamyl amino acid and L-amino acid, glycine, and β-alanine, and ATP in an aqueous medium, and whether or not a peptide is produced and accumulates in the aqueous medium by HPLC It can confirm by the method of analyzing by etc.
3. Microorganisms and transformants used in the production method of the present invention The microorganisms and transformants used in the production method of the present invention are not particularly limited as long as they are microorganisms and transformants capable of producing the protein of the present invention. However, the microorganism is preferably a microorganism belonging to the genus Clostridium , more preferably a microorganism belonging to Clostridium acetobutylicum , and more preferably Clostridium acetobutylicum ATCC824. And a transformant transformed with a DNA encoding the protein of the present invention.

Examples of the transformant transformed with the DNA encoding the protein of the present invention include a transformant obtained by transforming a host cell by a known method using a recombinant DNA containing the above DNA 2. Examples of host cells include prokaryotes such as bacteria, yeast, animal cells, insect cells and plant cells, preferably bacteria, more preferably microorganisms belonging to the genus Escherichia .
4). Preparation of DNA of the Present Invention The DNA of the present invention is a microorganism belonging to the genus Clostridium , preferably Clostridium acetobutylicum (for example) using a probe that can be designed based on the base sequence represented by SEQ ID NO: 2. Clostridium acetobutylicum ), more preferably Southern hybridization to the chromosomal DNA library of Clostridium acetobutylicum ATCC824, or a microorganism using a primer DNA that can be designed based on the nucleotide sequence represented by SEQ ID NO: 2, preferably It can be obtained by PCR [PCR Protocols, Academic Press (1990)] using a microorganism belonging to the genus Clostridium , more preferably a microorganism belonging to Clostridium acetobutylicum , more preferably a chromosomal DNA of Clostridium acetobutylicum ATCC824.

  In addition, the base sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 1 with respect to various gene sequence databases is 80% or more, preferably 90% or more, more preferably 94% or more, and still more preferably 98% or more. Particularly preferably, 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 can also be obtained.

The obtained DNA is cleaved as it is or with an appropriate restriction enzyme, and incorporated into a vector by a conventional method. After the obtained recombinant DNA is introduced into a host cell, a commonly used nucleotide sequence analysis method such as the dideoxy method [ Proc. Natl. Acad. Sci. USA, 74 , 5463 (1977)] or Applied Biosystems 3700 DNA Analyzer (Applied Biosystems), etc. Can be determined.

If the obtained DNA has a partial length as a result of determining the base sequence, the full-length DNA can be obtained by Southern hybridization or the like for a chromosomal DNA library using the partial length DNA as a probe. .
Furthermore, based on the determined DNA base sequence, 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.
Examples of vectors that incorporate the DNA of the present invention include pBluescript II KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18 , 6069 (1990)], pPCR-Script Amp (Stratagene), pT7Blue (Manufactured by NOVAGEN), pCR II (manufactured by Invitrogen), and 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 ATCC12435, Escherichia coli W1485, Escherichia coli W1485, 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. .

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell. For example, 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.
Examples of the transformant of the present invention obtained by the above method include Escherichia coli BL21 / pCAC1540, which is a microorganism having a recombinant DNA containing a DNA having the sequence represented by SEQ ID NO: 2. .
5. Method for producing transformant and microorganism used in the production method of the present invention Based on the DNA of the present invention, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein of the present invention is prepared. To do. In addition, a transformant with an improved production rate of the protein can be obtained by substituting the base sequence of the portion encoding the protein so that the codon is optimal 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.

As the expression vector, a vector that can replicate autonomously 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.
When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant DNA having the DNA of the present invention can replicate autonomously in the prokaryotic organism, and at the same time, the promoter, the ribosome binding sequence, the DNA of the present invention, the transcription termination. It is preferably a recombinant DNA composed of a sequence. A gene that controls the promoter may also be included.

The expression vectors include pBTac1, pBTac2 (boehringer Mannheim), pHelix1 (Roche Diagnostics), pKK233-2 (Amersham Pharmacia Biotech), pSE280 (Invitrogen), pGEMEX- 1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pET-3, pET-21d (+) (all manufactured by Novagen), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol Chem., 48 , 669 (1984)], pLSA1 [Agric. Biol. Chem., 53 , 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82 , 4306 (1985)], pBluescript II SK (+), pBluescript II KS (-) (Stratagene), pTrS30 (prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)), pTrS32 [Escherichia coli JM109 / pTrS32 (FERM BP-5408 ))], PPAC31 (WO 98/12343 pamphlet), pUC19 [Gene, 33 , 103 (1985)], pSTV28 (Takara Bio), pUC118 (Takara Bio), Examples thereof include pPA1 (Japanese Patent Laid-Open No. 63-233798).

Any promoter can be used as long as it functions in a host cell such as Escherichia coli. For example, trp promoter (P _trp), cited lac promoter (P _lac), P _L promoter, P _R promoter and 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. In addition, 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.

Furthermore, 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.
It is preferable to use 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 (for example, 6 to 18 bases).

In the recombinant DNA in which the DNA of the present invention is bound to an expression vector, a transcription termination sequence is not necessarily required, but it is preferable to place the transcription termination sequence immediately below the structural gene.
An example of such recombinant DNA is pCAC1540.
Prokaryotic used as host cells, Escherichia (Escherichia) genus Serratia (Serratia) genus Bacillus (Bacillus) genus Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus, genus Brevibacterium (Microbacterium) , Pseudomonas (Pseudomonas) genus Agrobacterium (Agrobacterium) genus, belonging to the genus Alicyclobacillus (Alicyclobacillus), Anabaena (Anabena) genus Anashisutisu (Anacystis) genus Arthrobacter (Arthrobacter) genus Azotobacter (Azotobacter) genus, Kuromachiumu (Chromatium) genus, Erwinia (Erwinia) genus, Methylobacterium (Methylobacterium) genus, Phormidium (Phormidium) genus Rhodobacter (Rhodobacter) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Rodosupiriumu (Rhodospirillum) genus, Se Desumusu (Scenedesmus) genus Streptomyces (Streptomyces) genus, Shinekokkasu (Synechoccus) genus, microorganisms belonging to Zymomonas (Zymomonas) species, such as, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 , Escherichia coli DH5α, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, NY E. coli MP347, Escherichia coli NM522, Escherichia coli BL21, Bacillus subtilis (Bacillus subtilis) ATCC33712, Bacillus megaterium (Batillus megaterium), Brevibacterium Lee Mario film (Brevibacterium immariophilum) ATCC14068, Burebiba Agrobacterium Sacca Lori tee cam (Brevibacterium saccharolyticum) ATCC14066, Brevibacterium flavum (Brevibacterium flavum) ATCC14067, Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869, Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, Corynebacterium glutamicum ATCC14297, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC6872, Corynebacterium acetamide A Sid film (Corynebacterium acetoacidophilum) ATCC13870, micro Corynebacterium ammoniagenes film (Microbacterium ammoniaphilum) ATCC15354, Serratia Fikaria (Serratia ficaria), Serratia・ Fontacola ( Serratia fonticola ), Serratia liquefaciens ( Serratia liquefaciens ), Serratia marcescens ( Serrati a marcescens), Pseudomonas sp. (Pseudomonas sp.) D-0110 , Agrobacterium radiobacter (Agrobacterium radiobacter), Agrobacterium Rizzo jeans (Agrobacterium rhizogenes), Agrobacterium ruby (Agrobacterium rubi), Anabaena, Shirindorika (Anabaena cylindrica), Anabaena-Doriorumu (Anabaena doliolum), Anabaena floss Aqua (Anabaena flos-aquae), Arthrobacter Ore' sense (Arthrobacter aurescens), Arthrobacter citreus (Arthrobacter citreus), Arthrobacter glob folder miss (Arthrobacter globformis), Arthrobacter hydro carbon glutamates Mika scan (Arthrobacter hydrocarboglutamicus), Arthrobacter Misorensu (Arthrobacter mysorens), Arthrobacter Nicotiana (Arthrobacter nicotianae) Arthrobacter paraffineus (Arthrobacter paraffineus), Arthrobacter proto folder Mie (Arthrobacter protophormiae), Arthrobacter Rose opal Rafi eggplant (Arthrobacter roseoparaffinus), Arthrobacter sulphureus (Arthrobacter sulfureus), Arthrobacter urea Fashiensu (Arthrobacter ureafaciens), Kuromachiumu-Buderi (Chromatium buderi), Kuromachiumu-Tepidamu (Chromatium tepidum), Kuromachiumu-Binosamu (Chromatium vinosum), Kuromachiumu-Wamingi (Chromatium warmingii), Kuromachiumu-Furubiatatire (Chromatium fluviatile), Erwinia Uredobara (Erwinia uredovora), Erwinia Karotobara (Erwinia carotovora), Erwinia ananas (Erwinia ananas), Erwinia Herikora (Erwinia herbicola), d Binia-Pankutata (Erwinia punctata), Erwinia terreus (Erwinia terreus), Methylobacterium-Rodeshianamu (Methylobacterium rhodesianum), Methylobacterium-exo torque Enns (Methylobacterium extorquens), Phormidium sp (Phormidium sp.) ATCC29409, Rhodobacter Kapusuratasu (Rhodobacter capsulatus), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rod Pseudomonas Burasuchika (Rhodopseudomonas blastica), Rod Pseudomonas Marina (Rhodopseudomonas marina), Rod Pseudomonas pulse tris (Rhodopseudomonas palustris), Rodosupiriumu-Riburamu ( Rhodospirillum rubrum ), Rhodospirillum salexigens , Rhodospirillum salinarum , Streptomyces Vinegar ambofaciens (Streptomyces ambofaciens), Streptomyces aureofaciens (Streptomyces aureofaciens), Streptomyces aureus (Streptomyces aureus), Streptomyces Funjishidikasu (Streptomyces fungicidicus), Streptomyces Guriseokuromoge Eggplant ( Streptomyces griseochromogenes ), Streptomyces griseus ( Streptomyces griseus ), Streptomyces lividans , Streptomyces olivogriseus , Streptomyces rameus ( Streptomyces rameus ( Streptomyces rameus ) Tanashienshisu (Streptomyces tanashiensis), Streptomyces Binaseusu (Streptomyces vinaceus), Zymomonas mobilis (Zymomonas mobilis), etc. can be mentioned .

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell. For example, 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.
When using a yeast strain as a host cell, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 etc. can be used as an expression vector, for example.

Any promoter may be used as long as it functions in yeast strains. For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter And promoters such as MFα1 promoter and CUP 1 promoter.
As the host cell, 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), it is possible to increase the Candida utilis (Candida utilis) and the like.

As a method for introducing recombinant DNA, any method for introducing DNA into yeast can be used. For example, electroporation method [Methods Enzymol., 194 , 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 81 , 4889 (1984)], lithium acetate method [J. Bacteriol., 153 , 163 (1983)] and the like.
When animal cells are used as hosts, 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, etc. Can do.

  Any promoter can be used as long as it functions in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter or metallothionein promoter, retrovirus Promoters, heat shock promoters, SRα promoters, and the like. In addition, 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.
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, COS-7, and the like.

As a method for introducing recombinant DNA, any method for introducing DNA into animal cells can be used. For example, electroporation [Cytotechnology, 3 , 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA, 84 , 7413 (1987)], Virology, 52 , 456 (1973), and the like.

When using insect cells as a host, for example, 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 recombinant virus is further infected into insect cells to produce proteins. it can.
Examples of gene transfer vectors used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.

As the baculovirus, for example, an autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
As 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 the 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)].

When plant cells are used as host cells, 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, and examples thereof include cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter and the like.

Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.
As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into plant cells. For example, a method using Agrobacterium (JP 59-140885, JP 60-70080, WO94 / 00977 pamphlet), electroporation method (Japanese Patent Laid-Open No. 60-251887), method using particle gun (gene gun) (patent 2606856, patent 2517813), etc. Can give.
6). Production method of the protein of the present invention 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.
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.

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.

As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
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 hours to 7 days. During the culture, 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.

Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
When culturing a microorganism transformed with an expression vector having an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic is used when a microorganism transformed with an expression vector using the trp promoter is cultured. An acid or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host, 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. can be used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and the presence of 5% CO ₂ .
Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.
As a medium for culturing a transformant obtained using insect cells as a host, commonly used 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 can be used.

Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7, 25 to 30 ° C.
Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.
A transformant obtained using a plant cell as a host can be cultured as a cell or differentiated into a plant cell or organ. As a medium for culturing 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 performed under conditions of pH 5-9 and 20-40 ° C. for 3-60 days.
Moreover, you may add antibiotics, such as kanamycin and a hygromycin, to a culture medium as needed during culture | cultivation.
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.

When the protein of the present invention is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. [J. Biol. Chem., 264 , 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad Sci. USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], or by applying the method described in JP-A-5-336963, WO94 / 23021 pamphlet, etc. The protein can be actively secreted outside the host cell.

That is, by using a gene recombination technique, 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.
Further, according to the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

Furthermore, by redifferentiating the cells of the transgenic animal or plant, the animal individual (transgenic non-human animal) or plant individual (transgenic plant) into which the gene has been introduced is created, The protein of the invention can also be produced.
When 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. Thus, the protein can be produced.

Examples of the method for producing the protein of the present invention using an animal individual include known methods [Am. J. Clin. Nutr., 63 , 639 (1996), Am. J. Clin. Nutr., 63 , 627 ( 1996), Bio / Technology, 9 , 830 (1991)], a method for producing the protein of the present invention in an animal constructed by introducing a gene.
In the case of an animal individual, for example, 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), eggs and the like. Any promoter can be used as long as it functions in animals. For example, a casein promoter, β casein promoter, β lactoglobulin promoter, whey acidity, which is a mammary cell specific promoter, can be used. A protein promoter or the like is preferably used.

As a method for producing the protein of the present invention using a plant individual, for example, a transgenic plant introduced with a DNA encoding the protein of the present invention can be obtained 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.

As a method for isolating and purifying the protein of the present invention produced by using the transformant producing the protein of the present invention, a usual enzyme isolation and purification method can be used.
For example, when the protein of the present invention is produced in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, an ultrasonic crusher, a French press, a manton. Cells are disrupted with a Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.

  From the supernatant obtained by centrifuging the 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, Cation exchange chromatography using a resin such as Q-Sepharose FF (Amersham Biosciences), Resin such as butyl sepharose and phenyl sepharose Using a method such as hydrophobic chromatography using gel, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Obtainable.

In addition, 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.

When the protein of the present invention or a derivative such as a sugar modification product thereof is secreted extracellularly, the protein or a derivative such as a sugar modification product thereof 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.

Examples of the protein thus obtained include a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
In addition, 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. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], JP-A-5-336963, WO94 / 23021. In accordance with the method described in the pamphlet, the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

In addition, 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)] or can 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.

Based on the amino acid sequence information of the protein obtained above, the protein of the present invention is produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method) and the like. 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.
7). Process for Producing Peptide of the Present Invention A culture of the above-mentioned microorganism or transformant or a processed product of the culture or the protein of the present invention of 1 above and one or more amino acids or dipeptides and amino acids in an aqueous medium The peptide can be produced by being present, generating and accumulating the peptide in the medium, and collecting the peptide from the medium.
(1) Method for Producing Peptide Using the Protein of the Present Invention as an Enzyme Source In the production method of the present invention, when the protein of the present invention is used as an enzyme source, the protein of the present invention produced by the above method 6 can be used. it can. In addition, a microorganism having the ability to produce the protein of the present invention, preferably a microorganism belonging to the genus Clostridium , more preferably a microorganism belonging to Clostridium acetobutylicum , more preferably Clostridium acetobutylicum ATCC824 by the method described below. The protein of the present invention that can be obtained by the above-described isolation and purification method from the culture obtained by culturing can also be used.

  In the production method of the present invention, when the protein of the present invention is used as an enzyme source, the substrate is one or more, preferably 1 to 3, more preferably 1 or 2 amino acids, or a combination of a dipeptide and an amino acid. Can give. When the substrate is one or more amino acids, any amino acid may be used as long as it is an amino acid selected from the group consisting of L-amino acids, glycine and β-alanine. Examples of L-amino acids include L-alanine, L-glutamine, L-glutamic acid, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, and L-serine. , L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, L-6-diazo-5-oxo-norleucine, N-acetyl-L-cysteine and N-acetyl-L-α-amino Examples include butyric acid.

  When the substrate is a combination of a dipeptide and an amino acid, the dipeptide includes γ-L-glutamyl amino acid, γ-D-glutamyl amino acid, γ-L- (α-methyl) glutamyl amino acid, β-aminoglutaryl amino acid, γ- Examples thereof include dipeptides selected from the group consisting of L- (N-methyl) glutamylamino acid, γ-DL- (β-methyl) glutamylamino acid, and γ-DL- (γ-methyl) glutamylamino acid. γ-L-glutamyl amino acids include γ-L-glutamyl-L-cysteine, γ-L-glutamyl-L-α-aminobutyric acid, γ-L-glutamyl-LS-methylcysteine, γ-L-glutamyl- L-serine, γ-L-glutamyl-L-alanine, γ-L-glutamyl-L-norvaline, γ-L-glutamyl-L-hypoglycine, γ-L-glutamyl-β-cyanoalanine, γ-L- Examples include γ-L-glutamyl amino acid selected from the group consisting of glutamyl-Se-methylselenocysteine and N- (γ-L-glutamyl) amino-D-proline. Further, when the substrate is a combination of a dipeptide and an amino acid, any amino acid may be used as the amino acid as long as it is an amino acid selected from the group consisting of L-amino acid, glycine and β-alanine. Examples of L-amino acids include L-alanine, L-glutamine, L-glutamic acid, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, and L-serine. , L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, L-6-diazo-5-oxo-norleucine, N-acetyl-L-cysteine and N-acetyl-L-α-amino Examples include butyric acid.

As a more preferable substrate used in the above production method, γ-L-glutamyl-L-cysteine and an amino acid selected from the group consisting of L-alanine, glycine, L-methionine, L-serine, and L-cysteine are used. Combinations can be given.
One or more amino acids, or dipeptides and amino acids, which are substrates, can be supplied as a powder or as a solution in an aqueous medium for generating and accumulating peptides. In this aqueous medium, chemical synthesis, enzymatic synthesis It can also be supplied by synthesizing a substrate by a method, a biological synthesis method or the like. For example, γ-glutamylcysteine synthetase derived from Escherichia coli [EC6.3.2.2], L-glutamic acid, L-cysteine, and ATP are allowed to coexist in an aqueous medium for generating and accumulating peptides. L-glutamyl-L-cysteine can be supplied. The substrate may be supplied before the peptide is generated and accumulated, or at the same time as the peptide generation.

In the above production method, the protein of the present invention is added in an amount of 0.01 to 100 mg, preferably 0.1 to 10 mg, per 1 mg of dipeptide or one amino acid used as a substrate.
In the above production method, the dipeptide and amino acid used as substrates are added to the aqueous medium at the beginning or in the middle of the reaction so as to have a concentration of 0.1 to 500 g / L, preferably 0.2 to 200 g / L, respectively.

In the above production method, ATP can be used as an energy source, and ATP is used at a concentration of 0.5 mmol to 10 mol / L. ATP can be supplied as a powder or as a solution in an aqueous medium for generating and accumulating peptides. For example, ATP regeneration activity using a glycolytic system or polyphosphate kinase (ADP generated during peptide generation is converted to ATP). ATP can also be supplied by adding to the aqueous medium. Specifically, a method of adding cultured cells and a carbon source of Corynebacterium ammoniagenes [Biosci. Biotechnol. Biochem., 65 , 644 (2001)], a method of adding polyphosphate kinase and polyphosphate [J. Biosci. Bioeng ., 91 , 557 (2001)].

  The aqueous medium used in the above production method may be an aqueous medium having any component or composition as long as it does not inhibit the peptide formation reaction. For example, water, phosphate, carbonate, acetate, borate, Examples thereof include buffers such as citrate and Tris. Further, it may contain alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide.

The peptide formation reaction is carried out in an aqueous medium at pH 5 to 11, preferably pH 6 to 10, 20 to 50 ° C, preferably 25 to 45 ° C, for 2 to 150 hours, preferably 6 to 120 hours.
Peptides produced by the above method include peptides in which amino acids are linked by peptide bonds, or C-terminal amino acid residues and amino acids of dipeptides, preferably γ-L-glutamyl amino acids and L-amino acids, glycine and β-alanine An amino acid selected from the group consisting of -Proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-asparagine Acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, L-6-di Peptides in which amino acids selected from the group consisting of zo-5-oxo-norleucine, N-acetyl-L-cysteine, N-acetyl-L-α aminobutyric acid, glycine and β-Ala are linked by peptide bonds, more preferably , A tripeptide in which cysteine residues of γ-L-glutamyl-L-cysteine and amino acids selected from the group consisting of L-alanine, glycine, L-methionine, L-serine and L-cysteine are linked by peptide bonds Can do.
(2) Method for Producing Peptide Using Culture or Processed Product of Microorganism or Transformant as Enzyme Source As a culture of microorganism used as an enzyme source in the production of the present invention, the protein of the present invention is produced. Examples thereof include a culture obtained by culturing a microorganism by a culture method suitable for the growth of the microorganism. For example, when Clostridium acetobutylicum ATCC824 is used as the microorganism, NBRC medium No. 807 medium (polypeptone 15 g / L, yeast extract 5 g / L, glucose 5 g / L, sodium chloride 2.5 g / L, L-cysteine 0.5 g / L L, sodium thioglycolate 0.5g / L, resazurin 1mg / L, pH 7.0-7.2) and ATCC medium 1017 medium (Grand beef 500g / L, 1N sodium hydroxide 25mL / L, peptone 30g / L, yeast extra 5.0g / L, dipotassium hydrogen phosphate 5.0g / L, 0.025% resazurin solution 4.0mL / L, L-cysteine 0.5g / L, pH 7.0), etc. A culture obtained by anaerobic culture using a gas pack (manufactured by Nippon Becton Dickinson) for 10 days can be given. Examples of the culture of the transformant used as an enzyme source in the production method of the present invention include a culture obtained by culturing the transformant by the culture method described in 6 above.

  The processed product of the culture of the microorganism or transformant may be a concentrate of the culture, a dried product of the culture, a surfactant-treated product of the culture, a solvent-treated product of the culture, Enzyme-treated product, cells obtained by centrifuging or filtering the culture, dried product of the cells, freeze-dried product of the cells, surfactant-treated product of the cells, An enzyme source such as a solvent-treated product, an enzyme-treated product of the microbial cell, and an immobilized product of the microbial cell, which contains a living microbial cell having the same function as the culture of the microorganism, Examples thereof include an ultrasonically treated product, a mechanically ground treated product of the bacterial cells, and a crude enzyme extract obtained from the treated bacterial cells.

When using a transformant or a culture of microorganism or a processed product of a microorganism as an enzyme source, one or more amino acids used as a substrate, or a combination of a dipeptide and an amino acid may be the same compound as in (1) above. I can give you.
The amount of the enzyme source varies depending on the specific activity of the enzyme source and the like, but for example, 5 to 1000 mg, preferably 10 to 400 mg is added as a wet cell weight per 1 mg of dipeptide or one amino acid used as a substrate.

One or more amino acids or dipeptides and amino acids used as a substrate can be supplied in an aqueous medium in the same manner as in (1) above. Similar to (1) above, ATP can be present in an aqueous medium and used as an energy source.
As the aqueous medium, the above medium (1) can be used. In addition, the culture supernatant of the microorganism or transformant culture used for the enzyme source can also be used as the aqueous medium.

The reaction conditions for the peptide production reaction can be the same conditions as in (1) above.
Examples of the peptide produced by the above method include the same peptides as in (1) above.
In the production methods of (1) and (2) above, the peptide produced and accumulated in the aqueous medium can be collected by an ordinary method using activated carbon, ion exchange resin or the like, or extraction with organic solvent, crystallization, thin layer chromatography. Can be carried out by chromatography, high performance liquid chromatography and the like.

  Examples are shown below, but the present invention is not limited to the following examples.

Construction of CAC1540 gene expression strain SEQ ID NO: 2 present on the chromosomal DNA of Clostridium acetobutylicum ATCC824
The nucleotide sequence information of the CAC1540 gene that encodes a protein with unknown function (http://gib.genes.nig.ac.jp/single/index.php?spid=Cace_ATCC824, http: // Based on www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=15024486&itemID=4&view=gbwithparts), the homologous gene of CAC1540 gene was obtained from the chromosomal DNA of Clostridium acetobutylicum ATCC824 as follows.

First, Clostridium acetobutylicum ATCC824 strain was added to NBRC medium No.807 medium (polypeptone 15 g / L, yeast extract 5 g / L, glucose 5 g / L, sodium chloride 2.5 g / L, L-cysteine 0.5 g / L, thioglycol Sodium acid 0.5g / L, resazurin 1mg / L
PH 7.0-7.2) Inoculated to 10 mL and anaerobically cultured at 37 ° C. for 24 hours. 1 mL of the culture solution was inoculated into 250 mL of NBRC medium No. 807 medium, and statically cultured at 37 ° C. for 48 hours. Chromosomal DNA was prepared from cells collected by centrifugation using DNeasy Kit (Qiagen).

Using the 8905 type DNA synthesizer manufactured by Perseptive Biosystems, DNAs having the nucleotide sequences represented by SEQ ID NOs: 3 and 4 (hereinafter referred to as primer A and primer B, respectively) were synthesized. Primer A is obtained by adding a nucleotide sequence containing a Nco I recognition sequence at the 5 'end of the region containing the initiation codon of CAC1540 gene chromosomal DNA of Clostridium acetobutylicum ATCC824 strain. Primer B is obtained by adding a base sequence containing an Xho I recognition sequence to the 5 ′ end of a base sequence complementary to a DNA sequence containing the C-terminal amino acid sequence of the CAC1450 gene.

For amplification of the homologous gene fragment of the CAC1450 gene, PCR was performed using the above primer A and primer B and the chromosomal DNA of Clostridium acetobutylicum ATCC824 strain as a template. PCR was performed using 0.1 μg of chromosomal DNA, 0.5 μmol / L of each primer, 2 units of KOD plus DNA polymerase (Toyobo), 5 μL of KOD plus DNA polymerase × 10 buffer (Toyobo), 200 μmol / L each. Prepare 50 μL of reaction solution containing L dNTP (dATP, dGTP, dCTP and dTTP), heat at 95 ° C for 135 seconds, then at 95 ° C for 30 seconds, 52 ° C for 45 seconds, 68 ° C for 90 seconds Was repeated 25 times and further heated at 68 ° C. for 3 minutes.

1/10 volume of the reaction solution was subjected to agarose gel electrophoresis, and it was confirmed by PCR that an approximately 1.0 kb DNA fragment corresponding to the homologous gene fragment of the CAC1540 gene was amplified. The DNA fragment was purified using PCR DNA and Gel Band purification kit (Amersham Bioscience) and dissolved in 20 μL of TE.
When the base sequence of the DNA was determined by a known method, it was confirmed that it was the base sequence represented by SEQ ID NO: 2 encoding the amino acid sequence represented by SEQ ID NO: 1.

Next, 5 μL of the DNA solution obtained above is used, the DNA is cleaved with restriction enzymes Nco I and Xho I, DNA fragments are separated by agarose gel electrophoresis, and then GFX PCR DNA and Gel Band purification kit is used. Thus, a DNA fragment of about 1.0 kb containing the CAC1540 gene was recovered.
0.2μg of expression vectors pET-21d (+) was cleaved (manufactured by Novagen) with restriction enzymes Nco I and Xho I, the DNA fragments were separated by agarose gel electrophoresis, about 5.4kb in the same manner as described above DNA Fragments were collected.

The about 1.0 kb DNA fragment containing the CAC1540 gene obtained above and the about 5.4 kb DNA fragment of the expression vector pET-21d (+) obtained above were obtained at 16 ° C. using a ligation kit (Takara Bio Inc.) And then ligated for 1 hour.
Escherichia coli JM109 strain (manufactured by Takara Bio Inc.) is transformed with the reaction solution by a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)]. The body was applied to an LB agar medium containing 50 μg / mL ampicillin, and then cultured at 30 ° C. overnight.

By extracting a plasmid from the grown colonies of the transformant according to a known method and analyzing the structure using a restriction enzyme, the CAC1540 gene to which a sequence encoding a His tag sequence is added at the 3 ′ end is converted to T7. It was confirmed that pCAC1540, an expression vector linked downstream of the promoter, was obtained (FIG. 1).
Escherichia coli BL21 (DE3) strain (manufactured by Novagen) was transformed with pCAC1540 by a method using calcium ions, and the transformant was applied to an LB agar medium containing 50 μg / mL ampicillin, then 30 Incubate overnight at ° C.

  A plasmid was extracted from the grown transformant colony according to a known method, and the structure was analyzed using a restriction enzyme to confirm that pCAC1540 was retained.

Production of a protein having peptide synthesis activity Escherichia coli BL21 (DE3) (pushing Escherichia coli BL21 (DE3) / pCAC1540) containing pCAC1540 obtained in Example 1 was prepared in 3 mL of LB medium containing 50 μg / mL ampicillin. The test tube was inoculated and cultured with shaking at 30 ° C. for 16 hours. 100 μL of the obtained culture solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB medium. After culturing at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and the mixture was further cultured at 37 ° C. for 5 hours. The culture solution was centrifuged to obtain wet cells.

  The wet cells were disrupted by sonication, and then the His-tagged protein was purified from the supernatant obtained by centrifugation using HisTrap (His-tagged protein purification kit, manufactured by Amersham Biosciences). .

Production of tripeptides using His-tagged proteins 65 μg / L of the purified His-tagged protein obtained in Example 2, 50 mmol / L Tris-HCl buffer (pH 8.0), 12.5 mmol / L magnesium sulfate Then, a reaction solution consisting of 12.5 mmol / L ATP, 12.5 mmol / L γ-L-Glu-L-Cys and 12.5 mmol / L various L-amino acids was prepared and reacted at 37 ° C. for 19 hours. After completion of the reaction, the amount of phosphoric acid released in the reaction solution was quantified using Determiner L IP (manufactured by Kyowa Medex) to confirm the progress of the tripeptide formation reaction, and the reaction product was analyzed using MALDI-TOFMS. The production of tripeptides shown in Table 1 was confirmed.

  As shown in Table 1, the protein of the present invention has an activity of generating various peptides by linking the carboxyl group at the α-position of an amino acid, or the carboxyl group at the C-terminal of a dipeptide and the amino group of the amino acid by a peptide bond. I understood.

  According to the present invention, various peptides can be produced.

SEQ ID NO: 3 Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 4-Description of Artificial Sequence: Synthetic DNA

Claims (14)

The protein according to any one of [1] to [3] below.
[1] A protein having the amino acid sequence represented by SEQ ID NO: 1 [2] The amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and peptide synthesis 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 peptide synthesis activity The DNA according to any one of [1] to [3] below.
[1] DNA encoding the protein according to claim 1
[2] DNA having the base sequence represented by SEQ ID NO: 2
[3] DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having peptide synthesis activity A recombinant DNA containing the DNA according to claim 2. A transformant having the recombinant DNA according to claim 3. The transformant according to claim 4, wherein the transformant is a transformant obtained using a microorganism as a host. The transformant according to claim 5, wherein the microorganism is a microorganism belonging to the genus Escherichia . The method for producing a protein according to claim 1, wherein microorganisms capable of producing the protein according to claim 1 are cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture. The method for producing a protein according to claim 7, wherein the microorganism capable of producing the protein according to claim 1 is the transformant according to any one of claims 4 to 6. A culture of a microorganism having the ability to produce the protein according to claim 1 or a processed product of the culture or the protein according to claim 1 and one or more amino acids or amino acids and dipeptides in an aqueous medium, A method for producing a peptide, wherein the peptide is produced and accumulated in the medium, and the peptide is collected from the medium. The method for producing a peptide according to claim 9, wherein the amino acid is an amino acid selected from the group consisting of L-amino acid, glycine, and β-alanine. L-amino acid is L-alanine, L-glutamine, L-glutamic acid, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L- Threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4 From -hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, L-6-diazo-5-oxo-norleucine, N-acetyl-L-cysteine and N-acetyl-L-α aminobutyric acid The method for producing a peptide according to claim 10, wherein the peptide is an L-amino acid selected from the group consisting of: Dipeptide is γ-L-glutamyl amino acid, γ-LD glutamyl amino acid, γ-L- (α-methyl) glutamyl amino acid, β-aminoglutaryl amino acid, γ-L- (N-methyl) glutamyl amino acid, γ-DL- The method for producing a peptide according to claim 9, wherein the peptide is a dipeptide selected from the group consisting of (β-methyl) glutamylamino acid and γ-DL- (γ-methyl) glutamylamino acid. γ-L-glutamyl amino acid is γ-L-glutamyl-L-cysteine, γ-L-glutamyl-L-α-aminobutyric acid, γ-L-glutamyl-LS-methylcysteine, γ-L-glutamyl-L- Serine, γ-L-glutamyl-L-alanine, γ-L-glutamyl-L-norvaline, γ-L-glutamyl-L-hypoglycine, γ-L-glutamyl-β-cyanoalanine, γ-L-glutamyl- The method for producing a peptide according to claim 12, which is a γ-L-glutamyl amino acid selected from the group consisting of Se-methylselenocysteine and N- (γ-L-glutamyl) amino-D-proline. The method for producing a peptide according to any one of claims 9 to 13, wherein the microorganism having the ability to produce the protein according to claim 1 is the transformant according to any one of claims 4 to 6. .

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