JPH0740926B2 - Method for producing transformant and peptide precursor protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention utilizes transformant DNA technology to obtain transformants and peptides useful as precursor proteins for obtaining peptide precursors containing a plurality of peptides. Manufacturing method.

[Problems to be Solved by Conventional Techniques and Inventions] Currently, many peptide hormones having physiological activity are known, and needs for them as pharmaceuticals or reagents are increasing. In recent years, it has become possible to produce peptides by microorganisms using recombinant DNA technology. In this recombinant DNA technology, usually, a peptide precursor gene in which a gene encoding a peptide and a gene encoding another protein are linked is expressed in Escherichia coli as a host, and an enzyme or The target peptide is cut out by the action of a drug.

However, when a peptide precursor is produced using Escherichia coli as a host, there are the following problems.

(1) When Escherichia coli is used as a host, since endotoxin is produced, the endotoxin must be removed from the product.

(2) In many cases, the peptide precursor produced in the Escherichia coli cell is usually an insoluble mass called an inclusion body. To recover the peptide precursor from the inclusion body, urea or the like is used. The step of solubilizing with the solubilizing agent is required. The solubilization is particularly important when the peptide is cleaved from the peptide precursor by an enzymatic action or the like. However, this step is rather complicated and the solubilization efficiency is low. For example, when bovine growth hormone was produced by Escherichia coli, and the produced inclusion body was solubilized with urea, it was reported that the solubilization rate was 10%, and the rest remained forming the inclusion body. [Sc
honer, RG, et al., BIO / TECHNOLOGY, 3,151-154 (198
Five)].

(3) When the peptide precursor is produced in the bacterial cells, it is necessary to purify the desired peptide precursor from the disrupted bacterial cells. In this case, many purification steps are required in order to separate the desired peptide precursor from proteins derived from various types of host cells present in E. coli cells.

On the other hand, when the peptide precursor is secreted and produced outside the cells, the amount of the contaminant protein derived from the host is smaller than when the peptide precursor is produced inside the cells, which is advantageous for the production of the desired peptide precursor. Therefore, the Bacillus bacterium, which has the property of secreting a large amount of secreted protein and has no pathogenicity and which has been widely used for industrial production of enzymes and amino acids, is used as a host for secretory production of peptide precursors. It is being considered to make them. Among the Bacillus bacteria, Bacillus subtilis has many genetic and biochemical findings.
In addition, it has been reported that the secretory ability is utilized to secrete and produce many heterologous gene products using Bacillus subtilis as a host. For example, α of Bacillus subtilis
-Mice using the amylase gene-β-interferon secretion [Yamane, K. et al., In JAHoch and P. Se
tlow (ed.), Molecular biology of microbaial differ
entiation, American Society for Microbiology, Washi
ngton, DC117-123 (1985)], secretion of protein A of Staphylococcus aureus using the α-amylase gene of Bacillus amyloliquefaciens [St.
ephen R. Fahnestock, et al., Applied and Euvironmenta
l Microbiology, Feb.379-384 (1987)], secretion of β-lactamase of Escherichia coli using subtilisin gene [Sui-Lam Wang., et al., Journal of Bacteriology, Vo
l.168, No.2, Nov.1005-1009 (1986)] and human-α
-Secretion of interferon [Palva. I. et al., Gene, 22, 2
29-235 (1983)] and the like have been reported. As seen in these reports, a protein derived from a certain kind of prokaryote is efficiently secreted and produced using Bacillus subtilis as a host. For example, in the case of protein A of Staphylococcus aureus, a secretory production of about 1 g / is observed.

However, in the case of the secretion of eukaryote-derived mouse-β-interferon or human-α-interferon, its secretory production amount is small. For example, human-α-
In the case of interferon, its secretory production is 500 μg
/ It is about.

In addition, regarding secretory production of a peptide having a small number of amino acids or a precursor thereof, an atrial diuretic hormone (human atrial natriuretic α-factor (hAN) consisting of 25 amino acids using the subtilisin gene of Bacillus subtilis is used.
F)] has been reported [Lin-Fa Wang., Et
al., Gene, 69, 39-47 (1988)]. However, also in this case, the secretory production amount is as low as about 500 μg /. The main causes of this are considered to be inhibition of peptide secretion and degradation by a protease secreted by Bacillus subtilis.

These means that when Bacillus subtilis is used as a host, it is not easy to secrete and produce eukaryote-derived proteins or peptides outside the cells. In addition, there is a possibility that the C-terminal side of all or part of the secretory produced protein or peptide molecule may be decomposed by the protease derived from the host. Therefore, it is difficult to secrete and produce a peptide having a complete amino acid sequence by a microorganism such as Bacillus subtilis.

The object of the present invention is to eukaryote-derived peptides,
It is intended to provide a transformant useful for efficiently secreting and producing a peptide having a complete amino acid sequence in a large amount outside a cell.

Another object of the present invention is to provide a method for producing a peptide precursor protein, which can efficiently obtain a peptide having a complete amino acid sequence as a peptide precursor in a large amount.

[Structure of the Invention] In order to achieve the above-mentioned object, the present inventors have conducted extensive studies and as a result, have found that a gene encoding a protein secreted by a host microorganism to the outside of a bacterial cell is linked with a plurality of peptide-encoding genes. When a fragment and vector DNA are ligated and the resulting plasmid transforms a specific host microorganism having low protease productivity, the transformed strain contains a plurality of peptides having a complete amino acid sequence. The present invention was completed by discovering that a peptide precursor protein can be efficiently produced in a large amount outside the bacterial cell. That is, the present invention provides a gene encoding a protein secreted by a host microorganism to the outside of a microbial cell, a gene fragment in which a plurality of gene encoding a peptide are linked, and a plasmid in which a vector DNA is linked to a host microorganism. A transformed transformant, wherein the host microorganism lacks the ability to produce alkaline protease and neutral protease, and the protease activity is 3% or less of that of the wild strain.
Provided is a transformant that is a host microorganism into which the spoOAΔ677 mutant gene has been introduced.

The present invention also provides a method for producing a peptide precursor protein, which comprises culturing the transformant and secreting a peptide precursor protein containing a plurality of peptides to the outside of the microbial cell.

Hereinafter, the present invention will be described in detail.

The plasmid used in the present invention contains a gene encoding a protein secreted by the host microorganism outside the cell. The protein may be any protein that functions as a carrier and is secreted by the host microorganism to the outside of the microbial cell. A preferred protein is a bacterium of the genus Bacillus, especially Bacillus subtilis.
Is a protein secreted by. Bacillus subtilis is highly safe and secretes a large amount of protein outside the cells.

The protein secreted by the Bacillus bacterium outside the cell includes a protein originally secreted by the Bacillus bacterium, for example, α-
Not only amylase, neutral or alkaline protease, penicillinase, cellulase and the like, but also proteins of different organisms, for example, protein A derived from Staphylococcus aureus are included. Preferred proteins include protein A above.

Hereinafter, protein A will be described.

Protein A has a promoter region involved in gene expression and a ribosome binding site. The promoter region is derived from, for example, S. aureus 8325-4 strain [Uhren et al., J. Biol. Chem., 259.1695 (1984)],
It may be a promoter having a hairpin structure at positions -131 to -120 and a palindromic sequence at positions -151 to -138, but a preferred promoter region is proposed by the applicant in JP-A-63-245677. The region represented by the selected nucleotide sequence. This promoter region corresponds to the −300 to −th sequence, and its characteristic is that the above-mentioned S. aureus
This is a promoter region derived from the 8325-4 strain, and the hairpin structure and palindromic sequence do not exist.

A gene encoding an S region involved in secretion is present downstream of the promoter region (base sequence 1 to 108), and a structural gene is located downstream of the secretory sigwar region (base sequence 109 to 1524). Exists. The structural gene of protein A has E (109-276th nucleotide sequence), D (277
~ 459th base sequence), A (460 to 633rd base sequence), B (634 to 807th base sequence), C (808 to 981th base sequence), and X (982 to 1524th base sequence) Sequence) consisting of 6 units, from the N-terminal,
Combined in the above order. The 1525 to 1552th nucleotide sequence is a non-coding portion, and the 1525 to 1527th nucleotide has a termination codon TAA. The E region is a unit existing at the N-terminus of protein A, and has a relatively small ability to bind to immunoglobulin G (IgG). D, A, B,
The C region and the C region each have a strong binding ability to the Fc portion of 1 gG, and the X region is a unit having a function of binding the protein A molecule to the cell wall of S. aureus.

Thus, protein A has five repeating regions that specifically bind to the Fc region of IgG and a region that binds to the cell wall. Utilizing this fact, the peptide of interest can be linked after the region that specifically binds to the Fc region, expressed as a fusion protein, and then simply purified by affinity chromatography. Therefore, the gene encoding the protein is a region having the ability to bind to the Fc portion of IgG in the protein A, particularly up to the 440th amino acid in the thermogenic protein of protein A, that is, the structural gene of protein A. 44 of
A is the 0th amino acid (excluding the signal peptide)
The region up to sp is preferred. In addition, the protein is up to the 187th amino acid in the thermogenic protein of protein A,
That is, the region up to Leu which is the 187th amino acid (excluding the signal peptide) in the protein A structural gene is preferable.

In addition, as described above, protein A of Staphylococcus aureus
Contains a promoter, a ribosome binding site, a secretory signal, and a protein-encoding region involved in gene expression. Therefore, the plasmid having protein A introduced therein has a secretory expression ability, and when a peptide-encoding gene is ligated after a protein-encoding region, a peptide consisting of a protein and a peptide is secreted as a precursor protein. Express.

The size of the protein may be the whole protein molecule or a part thereof as long as it functions as a carrier.

The gene encoding the protein may be of biological origin or may be chemically synthesized. The gene may encode the whole protein or a part thereof, depending on the size of the protein.

The feature of the plasmid used in the present invention is that it includes a tandem-type gene in which a plurality of genes encoding the target peptide are linked to the gene encoding the protein.

The peptide is not particularly limited as long as it is composed of natural amino acids. Specific examples of the peptide include, for example, Vasoactintestinal polypeptide [Vasoacti
ve Intestinal Polypeptide (VIP)], essential diuretic hormone (ANP), insulin, gastrin, various opioid peptides, epidermal growth factor, endothelin, substance P, calcitonin, insulin-like growth factor I, II,
Examples thereof include physiologically active peptides such as galanin, motilin and vasopressin, and inhibitors such as hirudin, egulin C, secretory leukocyte-derived protease inhibitor and the like.
The peptides include human albumin, blood coagulation factor,
It also includes various differentiation-inducing factors such as lymphokines, nerve cell growth factors and hepatocyte regeneration factors, and proteins such as growth factors.

Preferred peptides include VIP, which consists of 28 amino acid residues and has pharmacological actions such as vasodilatory action and blood flow increasing action [Science 169, 1217 (1970)].
VIP is represented by the following amino acid sequence.

H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-T
yr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-L
ys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-OH The VIP represented by this amino acid sequence is that the 17th amino acid is Met instead of Leu, which is a prior art document [JP-A-1 -
296996 and Eur. J. Biochem. 178,343-350 (198
8)] different from VIP.

In addition, VIP has Gly-X at the C-terminus (wherein X is OH, Lys
-OH, Arg-OH, Lys-Arg-OH, or Arg-Lys-OH) is preferably added as a VIP precursor.

Genes encoding multiple peptides may be extracted from organisms or may be chemically synthesized. It should be noted that a gene that encodes a plurality of peptides of the same species in a continuous sequence is not generally known. Therefore, it is preferable to use a plurality of chemically synthesized genes as genes encoding a plurality of peptides. Further, the plurality of peptides may be composed of a plurality of peptides of the same kind, or may be composed of a plurality of peptides of different kinds.

The number of peptide-encoding genes linked to the protein-encoding gene may be 2 or more, but is usually about 2 to 20.

It is preferable that a spacer sequence (amino acid sequence) capable of being enzymatically or chemically cleaved is present between the plurality of genes encoding the peptide. The cleavable spacer sequence can be used to easily cut out and separate a peptide or a peptide-containing fragment from a peptide precursor. In addition, when a spacer sequence for cleavage is not required when isolating a peptide, it is not necessary to interpose the spacer sequence between a plurality of genes.

As the chemically cleavable spacer sequence, for example,
Met cleaved by cyanogen bromide [C-terminal side is cleaved. DVGoeddel, et al., Proc.Natl.Acad.Sci.USA76,10
6-110 (1979)], BNPS-Skatol and N-
Trp [C cleaved by chlorosuccinimide (NCS)
The end side is cut. Y.Saito, et al., J. Biochem. 101,1
23-134 (1987)], which is cleaved by an acid such as 70% formic acid. Asp-Pro [Asp-Pro is cleaved. Biochem.Biophy
s.Res.Commun., 40 , 1173 (1970)], Asn-Gly cleaved by hydroxyamine [Between Asn-Gly]
And so on.

The enzymatically cleavable spacer sequence is cleaved at the C-terminal side of Arg or Lys [Arg, Lys, which is cleaved by a trypsin-like enzyme such as trypsin or endoproteinase. J. Shine, et al., Nature, 285 , 456−461 (198
0)], Lysyl endopeptidase, endoproteinase Lys-C, and other Lys (the C-terminal side of Lys is cleaved. JP-A-61-275222), enzymes specific to acidic amino acids such as V8 protease. Glu and Asp (the C-terminal side of Glu and Asp is cleaved. Japanese Patent Publication No. 60-501391), and Ile-Glu-Gly-Arg (the C-terminal side is cleaved by blood coagulation factor Xa). JP Sho
61-135591), Gly-Pro-Arg and the like which are cleaved by thrombin (the C-terminal side is cleaved).
JP-A-62-135500), Kallikrei
Phe-Arg [C-terminal side which is cleaved by n) is cleaved. JP-A-62-248489H], Pro is cleaved by prolyl endopeptidase, and the C-terminal side is cleaved. Biochemistry, 16, 2942 (1977)], Glu-Gly-Arg (C-terminal side is cleaved, which is cleaved by urokinase, JP-A No. 2-100685) and the like.

Furthermore, enzymatically cleavable spacer sequences include the dipeptides Lys-Arg, Arg-Arg, Arg-Lys, Lys-Lys. A protease that specifically hydrolyzes these amino acids is derived from Escherichia coli.
OmpT Protease [Protease VII, K. Sugimoto, et a
L., J. Bacteriol. 170 , 5625-5632 (1988)], E-Protein derived from Salmonella typhimurium [J. Grodberg and JJDunn, J. Bacterio].
l. 171 , 2903-2905 (1989)], a protease involved in the biosynthesis of analgesic peptides [W. Demmer and K. Brand, Bioche.
m.Biophys.Res.Commun., 138 , 356-362 (1986)] and the like.

Examples of the protease that specifically hydrolyzes the spacer sequences Arg-Lys and N-terminal side of Arg-Arg include proteases involved in maturation of somatostatin [P. Glusch
ankof, et al., J. Biol. Chem. 262 , 9615-9620 (1987)]
And so on.

Furthermore, examples of the protease that specifically hydrolyzes the C-terminal side of the spacer sequences Lys-Arg and Arg-Arg include IRCM-Serine Protease 1
[JACromlish, et al., J. Biol. Chem, 261 , 10850-10858
(1986)], POMC-Converting Enzyme (Conv.
erting enzyme) [YPLoh, et al., J. Biol. Chem. 260 , 71
94-7205 (1985)], yeast Saccharomyces [Saccha
romyces, K.Mizuno, et al., Biochem.Biophys.Res.Commu
n. 144 , 807-814 (1987)], Kliberomyces sp.
uyveromyces), Sporobolomyces
s), genus Filobasidium, genus Hansenula, genus Issatchenki
a), Pichia, Rhodosporidium
sporidium), Saccharomycopsis
s) -derived protease (JP-A-1-191683, JP-A-2-49585) and the like.

Among these spacer sequences, Lys-Arg, Arg-Arg, or Pro-A capable of specifically hydrolyzing the C-terminal side with the yeast-derived basic amino acid residue pair-specific protease
rg is preferred.

The vector DNA in which the gene is incorporated to secrete the peptide precursor may be any DNA that can be replicated by the host microorganism, but is a DN of a plasmid that is replicable in Bacillus bacteria.
A is preferred. The vector, depending on the host secreting the protein outside the cells, a conventional vector, for example, plasmid pUB110 derived from Staphylococcus,
pC194, pBD64, pE194, pSAO501, pT127 and derivatives thereof. A preferred vector DNA is the DNA of plasmid pUB110. Bacillus subtilis carrying these plasmids are all available at the Bacillus Stock Center, University of Ohio (Address: 484, West 12th Avenue, Colu
mus Ohaio, 43210U.SA).

The plasmid of the present invention can be constructed by a conventional ligation technique, for example, a restriction enzyme method or a linker method in which a DNA cleaved with a restriction enzyme and a vector DNA are ligated with a ligase. That is, it is a control site for expression in a gene fragment in which a gene encoding the protein and a plurality of genes encoding peptides are linked. By binding a promoter, a ribosome binding site and a secretion signal, and binding the obtained DNA fragment to vector DNA,
A plasmid can be constructed. When using the above-mentioned protein A containing a promoter, a ribosome binding site, a secretory signal and a protein-coding region,
Genes encoding promoters, ribosome binding sites, secretion signals and proteins need not be introduced into the vector.

Conventional gene manipulation methods can be used for constructing genes encoding a plurality of peptides and ligating each gene fragment. At that time, an E. coli host vector or the like can also be used. For example, the construction of a tandem-type gene will be described using VIP as an example. To construct a gene encoding multiple VIPs, the restriction enzyme T should be added to the 5'side of the VIP gene.
The existence of GACNNNGTC, which is the cleavage recognition base sequence of th111I, can be utilized. The fragment cleaved by the control enzyme Tth111I has protruding ends, but does not have a symmetric structure.
It is extremely useful as a cleavage point for introducing a plurality of peptide-encoding genes in a desired direction by utilizing the cleavage site by the restriction enzyme Tth111I. That is, as the VIP gene to be inserted, a gene having a cleavage site for the restriction enzyme Tth111I on the 5'side and a cleavage site for the restriction enzyme on the 3'side is synthesized. On the other hand, the plasmid into which the VIP gene has been introduced is cleaved with the restriction enzyme Tth111I, the 5'side phosphate residue is removed with alkaline phosphatase, and the VIP gene to be inserted is ligated with DNA ligase. Escherichia coli is transformed with the obtained vector, and the transformed strain is cultured to prepare a plasmid into which two VIP genes have been introduced. By repeating the above operation,
Theoretically, using the Tth111I cleavage site, V
Tandemization of the IP gene is possible, and a plasmid in which multiple VIP genes are linked can be prepared.

A gene encoding another peptide can be tandemized by adding a cleavage site recognized by the restriction enzyme Tth111I to the 5'side of a gene encoding another peptide and sequentially ligating in the same manner as described above.

The transformant of the present invention can be obtained by introducing the above plasmid into a host microorganism and transforming the host microorganism. As a host microorganism, Bacillus subtilis, which secretes proteins outside the cells, accumulates many genetic and biochemical findings, and is highly safe
Among these, microorganisms having reduced protease productivity outside the cells are used. When such a strain is transformed with the above plasmid, decomposition by a protease derived from the host can be significantly suppressed, and a large amount of peptide precursor can be efficiently obtained.

As a Bacillus subtilis having a low protease productivity, Bacillus subtilis Bacillus subtilis, which lacks the ability to produce alkaline protease and neutral protease and has a protease activity of 3% or less of the wild strain, has spoOA △ 67.
7 Use a host microorganism into which a mutant gene has been introduced. Examples of such strains include Bacillus subtilis 104H, as proposed by the present inventors in Japanese Patent Application No. 1-281440.
L strain [Biochem.Biophys.Res.Commun., 128; 601-606, (19
85)], in which the spoOAΔ677 mutant gene was introduced into Bacillus subtilis SP011 strain (Microtechnology Research Institute No. 10987), Bacillus subtilis
Subtilis DY-16 strain (Microtech Lab. No. 9488) spoOA △ 677
Bacillus subtilis SPL14 strain (Microtechnological Research Institute No. 10988) and the like into which a mutant gene has been introduced are included.

Transformation of a host microorganism with the above-mentioned plasmid is carried out by a conventional method, for example, the method of protoplast formation by Chang et al. [Chang, S., et al., Mol. Gen. Genet., 168, 111-115 (19).
79)] and so on.

Furthermore, in the production method of the present invention, the transformant is cultured to secrete a peptide precursor protein containing a plurality of peptides outside the cells. Cultivation of the transformant can be carried out according to conventional liquid culture as long as the peptide precursor can be secreted and expressed. That is, the transformant can be cultivated in a liquid medium containing a conventional component such as an inorganic salt, a carbon source, a nitrogen source, a growth factor component and the like by shaking culture or aeration stirring culture method. PH of medium
Is, for example, about 7 to 8. Cultivation can be carried out under the usual conditions employed for culturing microorganisms, for example, a temperature of 15 to 45 ° C, preferably 25 to 40 ° C, and a culturing time of about 6 to 60 hours. More specifically, for example, 100 ml of Medium A medium [Stephen
R. Fahnestock.et al., Applied and Environmental Mic
robiology, Feb.379-384 (1987)], inoculate the transformant, and incubate at 37 ° C for about 15 hours.
A large amount of a peptide precursor protein containing a plurality of peptides can be secreted outside the microbial cell.

By subjecting the culture supernatant to a separation / purification means, for example, affinity chromatography, the peptide precursor protein can be separated and purified from the culture medium and recovered. The peptide contained in the recovered peptide precursor protein has a complete amino acid sequence without being decomposed.

The target peptide can be obtained by cleaving the spacer sequence of the peptide precursor protein by a chemical or enzymatic action. When cleaving the spacer sequence, it is preferable to cleave the C-terminal side of the spacer sequence. In particular, it is preferable to specifically hydrolyze the C-terminal side of the spacer sequence with a yeast-derived basic amino acid residue pair-specific protease. Incidentally, the above-mentioned protease, because the spacer sequence Arg-Lys, Lys-Lys sequence is not cleaved, it is relatively easy to prepare a large amount because it is of yeast origin, generally high safety, etc., Particularly preferred.

It can be used alone if the protease specifically recognizes the spacer sequence and specifically hydrolyzes the C-terminus of the spacer sequence.

When (a) the protease specifically recognizes the spacer sequence and specifically hydrolyzes the N-terminus of the spacer sequence or between the spacer sequences, (b) the basic amino acid from the N-terminus of the spacer sequence The peptide can be obtained by treating the fusion protein in combination with an aminopeptidase that releases C. and / or a carboxypeptidase that releases a basic amino acid from the C-terminus.

It specifically recognizes basic amino acids, and C of the spacer sequence
A carboxypeptidase or an aminopeptidase that releases a basic amino acid from the terminal side or the N-terminal side can be used regardless of its origin as long as the enzyme has such specificity. Examples of carboxypeptidase and aminopeptidase include carboxypeptidase B
(EC3.4.17.2), carboxypeptidase E (enkephalin convertase), carboxypeptidase N (EC3.4.17.3), yscα [KEX1 gene product derived from Saccharomyces cerevisiae; A. Dmochowska, et al., C
ell, 50 , 573-584 (1987)], aminopeptidase B
(EC3.4.11.6) etc.

The molecular weight of the obtained peptide can be measured by a conventional method, for example, agarose gel electrophoresis, SDS-polyacrylamide gel electrophoresis, etc.
This can be done by analyzing the amino acid sequence.

[Effects of the Invention] The plasmid of the present invention and the transformant transformed with the plasmid efficiently secrete and produce a large amount of a peptide having a complete amino acid sequence, even if the peptide is of eukaryotic origin.

According to the production method of the present invention, a peptide having a complete amino acid sequence can be efficiently obtained in a large amount as a peptide precursor.

[Examples] Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples. The enzymes used in the examples were all manufactured by Takara Shuzo Co., Ltd., and the reaction was carried out under the conditions described in their specifications.

Example 1 Production of peptide precursor protein using Bacillus subtilis as a host (1) Construction of plasmid pMD200 Using Bacillus subtilis as a host, the protein A gene of Staphylococcus aureus was expressed, and the protein in the culture supernatant was expressed. The construction diagram of the plasmid pMD200 that secretes and produces A is shown in FIG.

Plasmid pDCP2411 containing protein A gene
63-245677) is a plasmid in which a protein A gene cloned from one strain of Staphylococcus aureus is ligated to a plasmid pUC118 capable of replicating in E. coli. How to obtain pDCP2411 and pDCP2411
The entire base sequence of the protein A gene contained in the above is described in detail in the above-mentioned JP-A-63-245677.

From the E. coli transformant containing this plasmid, the alkaline method [Sambrook., Et al., Molecule Cloning., 1, 33 (198
9)] was used to prepare pDCP2411.

pDCP2411 was cleaved with restriction enzymes EcoRI and BamHI, and the resulting DNA fragment of about 1.9 kb containing the protein A gene (hereinafter referred to as protein A gene fragment) was separated by agarose gel electrophoresis and then electroeluted. Elute with
Purified [Sambrook. Et al., Molecular Cloning, 6, 28
(1989)]. The electric protein A gene fragment contains a promoter of the protein A gene, a ribosome binding site, a signal sequence for secretion, and a structural gene of protein A.

Then, the vector pUB110 was digested with the restriction enzymes EcoRI and BamHI.
A DNA fragment of about 3.7 Kbkb generated by cutting with and (below, pUB110
The vector gene fragment) was separated by agarose gel electrophoresis in the same manner as above, and then eluted and purified by electroelution.

The protein A gene fragment and the pUB110 vector gene fragment were ligated using T ₄ DNA ligase, and the plasmid pMD20
Constructed 0.

(2) Construction of a plasmid containing a gene encoding VIP In order to construct the VIP gene, the corresponding gene codons are matched to the codon usage of Bacillus subtilis according to the amino acid sequence of VIP, and the following eight types of genes are ABI430A DNA. It was produced by a synthesizer.

Using T _4-DNA kinase were added to phosphate at the 5 'end of each synthetic DNA. In addition, the plasmid pUC19 was cleaved with restriction enzymes Xba I and Sph I, and DNA ligase was used to
Each synthetic DNA with phosphate added to the 5'end was labeled with pUC
A plasmid pMD321a containing the VIP-Gly gene was constructed by inserting between 196Xba I and Sph I. Figure 2 shows the plasmid pMD32
The construction drawing of 1a is shown.

The nucleotide sequence and amino acid sequence of the obtained synthetic gene portion of pMD321a are shown below.

This synthetic gene part is the recognition sequence I1eGluGlyA of blood coagulation factor Xa, which is present before the nucleotide sequence encoding VIP-Gly.
It is constructed from rg, Met cleaved by BrCN, and a gene encoding the protein C recognition sequence ThrIlepheThrPheArg.

In addition, downstream of the VIP-Gly gene, after two termination codons (TAATAG), the terminator of subtilisin of Bacillus subtilis [M.Honjo. Et al., J. Biotechnology, 2, 75-85 (198
5)] exists.

(3) Construction of a plasmid containing a gene encoding 5 molecules of VIP Gene encoding a gene of 5 molecules of VIP (hereinafter referred to as tandem VIP
FIG. 3 shows a construction diagram of a plasmid pMD321R5a containing a gene).

The restriction enzyme Tth11 was added to the 5'side of the VIP-Gly gene of pMD321a.
There is GACGCAGTC which is a cleavage recognition base sequence of 1I. On the other hand, as shown in FIG. 3, a Tth111I cleavage site was left on the 5'side of the VIP gene to be inserted, and a gene incapable of cutting on the 3'side was synthesized.

Further, since the tandemized VIP-Gly is cleaved and recovered, a basic residue pair-specific protease (Japanese Patent Laid-Open No. 2-4958).
The gene corresponding to Lys-Arg, which is the recognition sequence for cleavage according to the publication No. 5), was introduced, and the VIP-Gly-Lys-Arg gene for tandemization was synthesized as shown in FIG.

Then, pMD321a was completely cleaved with Tth111I, and this 5'phosphate group was removed by alkaline phosphatase.
Then, the VIP-Gly-Lys-A prepared for tandem was prepared.
The rg genes were mixed and ligated with DNA ligase, and then Escherichia coli JM109 strain was transformed.

A plasmid gene was prepared from the obtained ampicillin-resistant transformant, and a plasmid pMD321R2a in which two VIP-Gly genes were linked in tandem was constructed as desired.

The resulting pMD321R2a is a VIP-Gl with two tandem proliferates.
It has a unique Tth111I breakpoint near the 5'region of the y gene.

Then, pMD321R2a was cleaved with Tth111I, the 5 ′ region was dephosphorylated with alkaline phosphatase, mixed with VIP-Gly-Lys-Arg gene for tandemization, ligated with DNA ligase, and Escherichia coli JM109 strain was ligated. Transformed. A plasmid was prepared from the obtained transformant resistant to ampicillin to obtain a plasmid pMD321R3a in which three target genes were introduced in tandem. The same operation was further repeated to prepare pMD321R5a in which five VIP-Gly genes were linked in tandem via Lys-Arg.

(4) Construction of plasmid pMD500R5 FIG. 4 shows the construction of plasmid pMD500R5 which secretes a peptide precursor containing 5 molecules of VIP (hereinafter referred to as a peptide precursor) using Bacillus subtilis as a host.

After cutting the plasmid pMD200 with the restriction enzyme Pst I, D
Blunting Kit was used to blunt the ends, followed by digestion with the restriction enzyme Sph I, and a fragment of approximately 5.5 Kb was purified by agarose gel electrophoresis and used as a vector to incorporate the tandem VIP gene. did.

The tandem VIP gene to be incorporated into the vector was prepared by cutting the plasmid pMD321R5a with the restriction enzyme KpnI, blunting the ends with a DNA branching kit, and further cutting it with the restriction enzyme SphI to obtain a fragment of about 0.5 Kb. It was obtained by purification by acrylamide gel electrophoresis. This DNA fragment has a structure in which a tandem VIP gene is linked downstream of a gene encoding an amino acid sequence recognized by blood coagulation factor Xa and protein C. Also,
At the C-terminal of each VIP gene, Gl
The gene encoding y is linked. In addition, VIP-Gl
Between the gene encoding y, the peptide encodes the recognition sequence Lys-Arg of PBRS-protease, which is a type of basic amino acid residue-specific protease, in order to recover the peptide by cleaving VIP-Gly from the precursor protein. Gene has been inserted.

Then, a vector DNA and tandem VIP gene fragment T ₄
Ligation was used to construct the peptide precursor protein secretion plasmid pMD500R5. This pMD500R5 uses Bacillus subtilis as a host, and contains Arg-G containing the amino acid sequences corresponding to the recognition sequences of blood coagulation factor Xa and protein C as protein spacers 1 to 402 and a spacer.
ly-Ser-Ser-Arg-Val-Asp-Val-Ile-Glu-Gly-A
rg-Met-The-Ile-Phe-Thr-Phe-Arg, VIP biosynthetic precursor VIP-Gly as a target peptide, is a plasmid having a structure in which 5 molecules are linked via Lys-Arg.

(5) Production of peptide precursor using Bacillus subtilis as a host Method by Chang et al. [Chang, S., et al., Mol. Gen.G.
e .., 168, 111-115 (1979)], pMD500R5 was used to transform Bacillus subtilis SPL14 (FERM P-10988). Obtained transformant Bacillus subtilis
The amount of VIP in the peptide precursor protein secreted by SPL14 (pMD500R5) (Microtechnology Research Institute No. 11742) was measured by the following method.

First, Medium A medium containing 0.5M succinic acid 10
Using 0 ml, SPL14 (pMD500R5) was shake-cultured at 37 ° C. for 16 hours. After stopping the culture, phenylmethylsulfonyl fluoride (PMSF), which is a protease inhibitor, and EDTA were added so that the concentration was 10 mM, and the cells were removed by centrifugation at a temperature of 4 ° C. and 15000 rpm for 10 minutes.

After filtering the culture supernatant with a 0.22 μm filter, 2 ml of I
2 g of the supernatant of the obtained culture was injected into a column packed with gG-Sepharose gel (Pharmacia Co., Ltd.) using a syringe having a volume of 5 ml to purify the peptide precursor protein. Load the column with 20 ml TST buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.
After washing with 05% Tween20], 0.1M acetic acid 1
The peptide precursor protein was eluted with 0 ml (pH 3.0). The eluate was immediately neutralized with 1/2 volume of 0.2M ammonium bicarbonate solution. The obtained peptide precursor protein eluate was concentrated using a filter for ultrafiltration (molecular weight cutoff 10,000), and then fractionated by reverse phase high performance liquid chromatography under the following conditions.

Column TSK gel Phenyl 5pPW-RP Inner diameter 4.6 mm × 7.5 cm Flow rate 1.0 ml / min Solvent A: 0.05% trifluoroacetic acid B: 0.044% trifluoroacetic acid / 60% acetonitrile Gradient 15.0 to 38% / 46 min, 2 ml /% , VIP-Gly was quantified by the enzyme immunoassay according to the method described in "Enzyme Immunoassay" (edited by Eiji Ishikawa et al., Medical Institute).

First, rabbits were immunized with VIP to obtain anti-VIP serum.
From this anti-VIP serum, anti-VIP-IgG, anti-VIP- by the maleimide-hinge method described on pages 83-92 of "Enzyme Immunoassay".
F (ab ') ₂ , anti-VIP-peroxidase labeled-Fab' was prepared.

VIP-Gly was measured by the enzyme immunoassay according to the following procedure.

Anti-VIP-F (ab ') ₂ was adsorbed on an ELISA plate (96 wells) and blocked with 1% bovine serum albumin. VIP-Gly to which a test solution was added was bound to anti-VIP-F (ab ') ₂ adsorbed on a solid phase, washed, and then anti-VIP-peroxidase-labeled-Fab ₂ was added to this plate. VIP-Gly bound to a solid phase was sandwiched. After removing the free labeled antibody, 10 mM ortho-phenylenediamine (OP
D), 0.025% hydrogen peroxide, and a reaction solution containing 50 mM sodium acetate buffer (pH 5.0) were added, and the dye produced by the reaction of peroxidase was measured by absorption at a wavelength of 490 nm.

As a reference material, Applied Biosystems (AB
I) VIP-Gly was used, which was subjected to solid-phase synthesis with a peptide synthesizer manufactured by the same company, purified by reverse-phase high performance liquid chromatography, and whose amino acid composition was confirmed.

As a result, the peptide precursor protein chamber of interest was secreted and produced, and in ELISA, the protein amount was calculated from the absorption at the wavelength of 214 nm of the peak showing VIP activity, and the molecular weight of the VIP portion predicted from the nucleotide sequence was calculated. Therefore, VIP contained in the secreted peptide precursor protein is
It was 3.2 mg /.

Example 2 Production of Peptide Precursor Using Bacillus subtilis as Host The above Bacillus subtilis SPL14 was subjected to qualitative conversion in the same manner as in Example. Obtained transformant SP
The peptide precursor protein secreted by L14 (pMD500R5) was purified as follows.

First, SPL14 (pMD500R5) was permeabilized at 37 ° C. for 16 hours using Medium A medium 4 containing 0.5 M succinic acid.
After stopping the culture, PMSF and EDTA were added so that the concentration was 10 mM, and the cells were removed by centrifugation at 4 ° C. and 5000 rpm for 30 minutes to remove the cells. After filtering the obtained culture supernatant with a 0.22 μm filter, 3.61 ml of the culture supernatant was injected into a column packed with 300 ml of IgG-Sepharose gel (Pharmacia Co., Ltd.) at a flow rate of about 6 ml / min. The peptide precursor protein was purified. The column was washed with TST buffer 2.88 having the same composition as in Example 1 at a flow rate of 4 ml / min, and then 0.1M acetic acid (pH 3.0) was used at a flow rate of 4 ml / min. The protein was eluted. At this time, the eluate was immediately neutralized with a 1/2 volume of 0.2 M ammonium bicarbonate solution. The obtained peptide precursor protein eluate was concentrated using a filter of ultrafiltration (molecular weight cut 10,000), and the peptide precursor was fractionated under the following conditions.

Column Phenyl-5PWRP Inner diameter 21.5 mm × 15 cm Flow rate 6 ml / min Solvent A: 0.05% trifluoroacetic acid B: 0.044% trifluoroacetic acid / 60% acetonitrile Gradient 27.5-30% / 50 min, 120 ml /% And separated components The desired peptide precursor protein having a molecular weight of 64000 was obtained by freeze-drying. Further, when the amount of protein was calculated from the absorption at a wavelength of 214 nm in the same manner as in Example 1, about 37 mg of peptide precursor protein was obtained from the culture solution of 4.

Reference Example 1 Extraction of VIP-Gly-Lys-Arg from peptide precursor protein 100 μg of the separated peptide precursor protein was treated with a basic amino acid residue-specific protease (JP-A-2-49585).
Gazette) 0.1mU [1U is 50 mM Tris-HCl (pH 7.
0), 0.1% Lubrol PX, 0.5 mM CaCl ₂ in 0.1 mM Boc-Gln-Arg-Arg-MCA (Boc is t
-Butoxycarbonyl group, MCA is 4-methylcoumaryl-
7-amide), and 1 mMole AMC (7-amino-4-methyl-coumarin) was released in 1 minute under the condition of reacting with 50 mM Tris-HCl (pH
7.0), and reacted at 37 ° C. in 1 mM CaCl ₂ . Reaction time 6
The reaction solution at time was analyzed by reverse phase high performance liquid chromatography, two peaks that appeared were separated, and their amino acid sequences were analyzed using a protein sequencer. VIP-Gly-Lys-Arg, And VIP-Gly. From this, it has a complete amino acid sequence
It was found that a peptide precursor containing VIP was secreted and produced.

Reference Example 2 VIP-Gly was excised from a peptide precursor protein, and 100 μg of the peptide precursor protein was taken out, and a basic amino acid residue-specific protease (JP-A-2-4958) was used.
5) 0.1 mU (1 U means the same activity as above) and carboxypeptidase B (manufactured by Sigma) 10 mU [1 U is 25
1 mM Bz-Gly-Arg in mM Tris-HCl (pH 8.0)
(Bz represents a benzyloxycarbonyl group) and an enzyme activity of releasing 1 μmol of Arg per minute under the condition of reacting at 25 ° C.] and 50 mM Tris-HCl (pH 7.
0), and reacted at 37 ° C. in 1 mM CaCl ₂ . The reaction solution at a reaction time of 6 hours was analyzed by reverse phase high performance liquid chromatography, the two peaks that appeared were separated by reverse phase high performance liquid chromatography, and their amino acid sequences were analyzed by using a protein sequencer. VIP
-Gly matched perfectly. From this, it was revealed that the peptide precursor containing VIP having the complete amino acid sequence was secreted and produced.

[Brief description of drawings]

FIG. 1 is a construction diagram of plasmid pMD200, FIG. 2 is a construction diagram of plasmid pMD321a, FIG. 3 is a construction diagram of plasmid pMD321R5a, and FIG. 4 is a construction diagram of plasmid pMD500R5.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 125) (C12P 21/02 C12R 1: 125) (56) References JP-A-60-500480 (JP , A) JP-A-1-95798 (JP, A) JP-A-63-71195 (JP, A) JP-A-59-205996 (JP, A) Actually published 63-245677 (JP, U) International Publication 89 / 5857 (WO, A)

Claims (9)

[Claims] 1. A host microorganism is transformed by a plasmid in which a gene fragment in which a plurality of gene encoding a peptide are linked to a gene encoding a protein secreted by the host microorganism to the outside of the cell and a vector DNA are linked. The transformed transformant, wherein the host microorganism lacks the ability to produce alkaline protease and neutral protease and the protease activity is 3% or less of that of the wild strain, sp.
A transformant that is a host microorganism into which oOAΔ677 mutant gene has been introduced. 2. The transformant according to claim 1, wherein the protein is a protein secreted by a bacterium of the genus Bacillus. 3. The protein secreted by Bacillus bacteria is Staphylococcus aureus.
aureus) protein A. The transformant according to claim 2. 4. A peptide-encoding gene is a vasoactive intestinal polypeptide (Vasoctive).
The transformant according to claim 1, which comprises a base sequence encoding an Intestinal Polypeptide). 5. The transformant according to claim 1, wherein the vector DNA is a plasmid DNA capable of replicating in a bacterium of the genus Bacillus. 6. The transformant according to claim 5, wherein the plasmid capable of replicating in Bacillus bacteria is pUB110. 7. The host microorganism is Bacillus subtilis 104H.
Bacillus subtilis SP011 strain (Microtechnical Research Institute No. 10987) in which the spoOA △ 677 mutant gene was introduced into the L strain, and Bacillus subtilis SPL14 strain (small in which the spoOA △ 677 mutant gene was introduced into the Bacillus subtilis DY-16 strain The transformant according to claim 1, which is Koken Bacteria No. 10988). 8. The transformant is Bacillus subtilis (Ba
cillus subtilis) SPL-14 (PMD500R5)
11742) and the transformant according to claim 1. 9. A method for producing a peptide precursor protein, which comprises culturing the transformant according to claim 1 and secreting a peptide precursor protein containing a plurality of peptides to the outside of the microbial cell.