JPH0740924B2 - Method for producing transformant and fusion protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention uses vasoactive
The present invention relates to a transformant useful for obtaining a precursor of intestinal polypeptide (hereinafter abbreviated as VIP) and a method for producing a fusion protein.

[Problems to be Solved by Prior Art and Invention] VIP is known as a physiologically active peptide having a relatively small molecular weight. This VIP consists of 28 amino acid residues and has been isolated by Said and Mutt from porcine duodenal mucosa as a peptide having a pharmacological action such as vasodilator and its structure has been determined. [Sc
ience 169, 1217 (1970)]. This VIP is synthesized in neurons of the intestine and brain, relaxes the digestive tract and blood vessels, and promotes small intestinal secretion.

Currently, the development of a drug is being studied by utilizing the tracheal dilation effect and blood flow increasing effect of VIP.
The amount of secretion is extremely small and it is difficult to obtain a large amount from the living body.

On the other hand, since VIP is a peptide, it may be considered to be produced by a microorganism by a gene recombination method. However, when a peptide having a relatively small molecular weight is directly expressed by a microorganism, the produced peptide is decomposed by a protease derived from the host, so that it is difficult to efficiently obtain a large amount of VIP.

Regarding a peptide having a pharmacological action equivalent to that of human VIP, JP-A-1-296996 and Eur. J. Biochem., 178., 343
-350 (1988), it is proposed to produce a VIP precursor or a VIP analog as a fusion protein with β-galactosidase derived from Escherichia coli in E. coli as a host. In this method, the produced fusion protein does not secrete and expresses, but accumulates in the cells.

Therefore, an object of the present invention is to provide a transformant useful for efficiently producing a large amount of a fusion protein in which a carrier protein and a VIP precursor are linked via a spacer sequence, outside the cell.

Another object of the present invention is to provide a production method capable of efficiently obtaining a VIP precursor in a large amount.

[Structure of the Invention] As a result of intensive studies to achieve the above-mentioned object, the present inventors
As a fusion protein in which a VIP precursor is linked to a carrier protein via a spacer sequence, the protease productivity is low, and in the case of secretory expression from a specific transformed host microorganism, degradation of the VIP precursor by a host-derived protease It was found that the fusion protein can be suppressed and the fusion protein is produced in large amounts outside the cells, and the present invention was completed.

That is, the present invention provides a promoter, a ribosome binding site, a secretory signal and a region encoding a carrier protein involved in gene expression, and a gene encoding a vasoactive intestinal polypeptide precursor, chemically or A DNA fragment, which is linked via an enzymatically cleavable spacer sequence, is a transformant obtained by transforming a host microorganism with a fusion protein secretory expression plasmid bound to vector DNA. The present invention provides a transformant, which is a host microorganism in which a spoOAΔ677 mutant gene is introduced into Bacillus subtilis lacking the ability to produce alkaline protease and neutral protease and having a protease activity of 3% or less of that of a wild strain.

The present invention also provides a method for producing a fusion protein, which comprises culturing the transformant, secreting a fusion protein in which a carrier protein and a baoactive intestinal polypeptide precursor are linked via a spacer sequence, and collecting the fusion protein. I will provide a.

The carrier protein contained in the plasmid used in the present invention is not particularly limited, but it is preferable that it has a high expression level and is stable, and further that the expressed fusion protein can be easily separated and purified. Examples of such a carrier protein include β-lactamase derived from Escherichia coli, alkaline phosphatase, α-amylase derived from Bacillus subtilis, protein A derived from Staphylococcus aureus, and the like. Protein A derived from Staphylococcus aureus has been reported to be stably expressed in Escherichia coli and Bacillus subtilis [Proc. Nat. Acad. Sci. USA,
80,697 ~ 701, (1983); J. Bacteriol., 165,796 ~ 804, (1
986)]. In particular, protein A is immunoglobulin G (I
5 repeating regions that specifically bind to the Fc region of gG),
It has a region that binds to the bacterial wall. Utilizing this fact, the desired VIP precursor can be ligated after the region that specifically binds to the Fc region, expressed as a fusion protein, and then simply purified by affinity chromatography. Therefore, the carrier protein is preferably a region having an IgG binding ability in protein A of Staphylococcus aureus.

Hereinafter, the base sequence of protein A and the amino acid sequence corresponding thereto are shown in the following formula [II], and protein A will be described in more detail.

Protein A is 5% of the cell wall component of S. aureus
It specifically binds to the Fc portion of human, mouse, rabbit, and other mammalian IgG, but does not bind to human IgG3. Further, protein A has a promoter region involved in gene expression and a ribosome binding site. The promoter region is, for example, S. aureus 8325-4 strain [Uhren et al., J. Biol. Chem., 259, 1695 (1
984)] and has a hairpin structure at positions -131 to -120 and a palindromic sequence at positions -151 to -138, but a preferred promoter region is disclosed by the applicant. It is a region represented by the nucleotide sequence proposed in 63-245677. This promoter region corresponds to the -300 to 1st sequence in the above formula [II], and its characteristics are different from those of the promoter region derived from the S. aureus 8325-4 strain, and the hairpin structure and palindrome are the same. The point is that the sequence does 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 from the secretory signal region (base sequence 109 to 1524). Exists.

The structural gene of protein A has E (109-276th nucleotide sequence), D (277-459th nucleotide sequence), A (460-633 nucleotide sequence).
Th base sequence), B (634th to 807th base sequence), C
(808 to 981st nucleotide sequence), and X (982 to 1524th nucleotide sequence), consisting of 6 units.
From the end, they are linked in the above order. In addition, 1525 ~ 15
The 52nd base sequence is the uncoated portion and is 1525
The ~ 1527th TAA is a stop codon. The above 6 units are obtained by digestion with trypsin, and are E, D, A,
The amino acid sequences of the B and C units are highly similar. The E region is a unit existing at the N terminus of protein A, and has a relatively small ability to bind to IgG. D,
Each of the A, B, and C regions has a strong binding ability to the Fc portion of IgG, and the X region is a unit having a function of binding a protein A molecule to the cell wall of S. aureus. Therefore, the carrier protein has a region capable of binding to the Fc portion of IgG, particularly up to the 440th amino acid in the mature protein of Protein A, that is, the 440th amino acid (signal peptide in the structural gene of Protein A). (Excluding) is the region up to Asp. Further, the carrier protein is preferably a region up to the 187th amino acid in the mature protein of Protein A, that is, up to Leu which is the 187th amino acid (excluding the signal peptide) in the structural gene of Protein A. .

As mentioned above, protein A of Staphylococcus aureus
It contains promoters involved in gene expression, ribosome binding sites, secretory signals, and regions encoding carrier proteins. Therefore, the plasmid into which protein A has been introduced has the secretory expression ability, and when the gene encoding the VIP precursor is ligated via the spacer sequence after the region encoding the carrier protein, the carrier protein, the spacer sequence and VIP It secretes and expresses a fusion protein consisting of a precursor.

The region encoding the carrier protein and the gene encoding the VIP precursor are linked via a chemically or enzymatically cleavable spacer sequence (amino acid sequence). When the region encoding the carrier protein and the gene encoding the VIP precursor are linked with the spacer sequence, the VIP precursor can be easily cleaved and separated from the produced fusion protein.

As the chemically cleavable spacer sequence, for example,
Met cleaved by cyanogen bromide [Met is cleaved behind Met. J. Biophys. Chem., 237, 1856 (1962)], Asp-Pro chain cleaved by formic acid [Biochem.Biophys.Res.
Commun., 40, 1173 (1970)] and the like. Also,
Enzymatic methods include, for example, sequences recognized by Factor Xa.
Ile-Glu-Gly-Arg [The rear of Arg is cleaved. J. Biol.
Chem., 249,7782 (1974)], sequences recognized by thrombin
Gly-Pro-Arg [The back of Arg is cut off. Nature, 218,3
14 (1968)], a sequence Phe-Arg recognized by kallikrein.
[The back of Arg is cut off. Hemostasis, 7,76 (197
8)], Glu recognized by V8 protease [The rear of Glu is cleaved. Pro.Nat.Acad.Sci.USA, 69,3506 (1972)],
The rear of Pro [Pro which is recognized by prolyl endopeptidase is cleaved. Biochemistry, 16, 2942 (1977)] and the like. As a method for producing the fusion protein,
Japanese Patent Publication No. 60-500480 discloses that protein A is used as a carrier protein. However, there is no disclosure regarding secretory expression of VIP precursor as a fusion protein.

As the VIP precursor, a VIP precursor having an amino acid sequence in which glycine (Gly) is added to the C terminus of VIP is preferably used. This VIP precursor is represented by the following formula [I].

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-Gly-
X [I] (In the formula, X is OH, Lys-OH, Arg-OH, Lys-Arg-OH,
Alternatively, it represents Arg-Lys-OH. ) The VIP precursor represented by this amino acid sequence is that the 17th amino acid is Met instead of Leu, and the above-mentioned prior art [JP-A-1-296996 and Eur.J.Biochem.178,343-3].
50 (1988)].

The fusion protein binds a promoter, a ribosome binding site, and a secretory signal, which are control sites for expression, to a DNA in which a region encoding a carrier protein and a gene encoding a VIP precursor are linked via the spacer sequence. Then, by ligating the obtained DNA fragment to vector DNA, a fusion protein secretion expression plasmid can be obtained. When using the above-mentioned protein A containing a promoter, a ribosome binding site, a secretory signal and a carrier protein, it is not necessary to introduce the promoter, the ribosome binding site, the secretory signal and the carrier protein into a vector.

The vector may be a conventional vector, for example, depending on the host.
pUB110, ColE1, pSC101, RSF2124, pBR322, Ti plasmid, pTUB4 [Takeich, Y. et al. agric. Biol. Chem., 47, 159
(1983); Yamazaki, H. et al. J. Bacterial.156 (1), 32
7 (1983)] and the like, bacteriophage λ
, Phage, SV40 virus, etc. can be selected.

A DNA recombinant obtained by incorporating a DNA fragment into vector DNA is prepared by a conventional method, for example, a restriction enzyme method or a linker method in which DAN fragmented with a restriction enzyme and vector DNA are ligated with a ligase. it can.

The microorganism (transformant) of the present invention can be obtained by transferring the above fusion protein secretion expression plasmid into a host microorganism and transforming it.

As a host microorganism, Bacillus subtilis, which is highly safe and secretes proteins outside the cells
Among these, microorganisms having reduced protease productivity outside the cells are used. When such a strain is transformed with the above plasmid, degradation of the fusion protein by a protease derived from the host can be significantly suppressed, and VIP precursor can be efficiently obtained in a large amount.

As a Bacillus subtilis with 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 that of the wild strain, has spoOA △ 67.
7 Use a host microorganism containing copper with the mutant gene. Such strains include, for example, 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)] into which the spo OA △ 677 mutant gene was introduced.
Subtilis SP011 strain (Microtech Lab. No. 10987), Bacillus subtilis DY-16 strain (Microtech Lab. No. 9488) spoOA △ 6
77 Bacillus subtilis SPL14 strain (Mikken Kenkyubo No. 10988), etc., in which a mutant gene is introduced.

Transfer of the recombinant DNA into the host can be carried out by a conventional method such as a protoplasting technique or a cell fusion technique.

In the method for producing a fusion protein of the present invention, a transformant transformed with the fusion protein secretion expression plasmid is cultured. The feature of the production method of the present invention is that, due to the secretory signal region held by the fusion protein secretion expression plasmid, the fusion protein is secreted and expressed by the culture, and the degradation of VIP precursor by the protease derived from the host microorganism is suppressed. To do. Cultivation of the transformant can be carried out in a medium containing a conventional component such as an inorganic salt, a carbon source, a nitrogen source, and a growth factor component. A preferred medium is
It is a liquid medium. For the culture in the liquid medium, a shaking culture or an aeration-agitation culture method can usually be adopted. The pH of the medium is usually about 7-8. Culturing is carried out under the usual conditions adopted for culturing microorganisms, for example, a temperature of 15 to 45 ° C., preferably 25 to 40.
It can be carried out under conditions of a temperature of about 6 to 60 hours.

By chemically or enzymatically cleaving a fusion protein, that is, a fusion protein in which a carrier protein and a VIP precursor are linked via a spacer sequence, a large amount of VIP precursor can be efficiently obtained. The chemical or enzymatic cleavage can be performed by causing the fusion protein to act with a chemical agent or an enzyme that cleaves the spacer sequence.

VIP as a physiologically active peptide consisting of the following 28 amino acid residues: H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-T
yr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-L
The ys-Lys-Tyr-Leu- Asn-Ser-Ile-Leu-Asn-NH 2 in the case of production by utilizing a gene recombination technique, C
An amide compound having an amino group at the end cannot be obtained. VIP is a VIP precursor and amidating enzyme or carboxypeptidase B
And an amidating enzyme, and amination of the C-terminal is obtained. More specifically, the VIP precursor represented by the formula [I] is reacted with the amidating enzyme or the like to convert Asn-Gly-X (X is the same as above) at the C-terminal of the amino acid sequence into Asn-. VIP can be obtained by converting to NH ₂ .

[Effects of the Invention] The transformant of the present invention efficiently produces a large amount of a fusion protein containing a VIP precursor outside the cells.

In addition, the method for producing a fusion protein of the present invention can secrete and express the fusion protein while suppressing the degradation of the VIP precursor by the protease, and efficiently produce a large amount of the VIP 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 of use.

Example 1 Secretion expression of protein A-VIP-Gly fusion protein by Bacillus subtilis-1 (1) Preparation of plasmid pMD200 Plasmid pDCP2411 containing protein A gene
63-245677) (10 μg) with the restriction enzyme EcoRI.
Digestion with BamH I (20untis each) gave a 2.25 kb EcoR I / BamH I fragment containing the protein A gene. Next, this 2.25 kb EcoR I / BamHI fragment (1 μg) and plasmid
pUB110 EcoR I / BamH I long chain fragment (0.5 μg) was added to T ₄ DN
Bacillus Bacillus ligated using A ligase (100units)
s subtilis PLS1 strain (available from the Bacillus Genetic Stock Center of Ohio State University) was transformed by the protoplast method] Mol.Gen.Genet., 168,111-11
5, (1979)].

The obtained kanamycin resistant (Km ^r ) strain was mixed with 10 ml of LB medium (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.2).
(Kanamycin Km was contained at 10 μg / ml), and the mixture was cultured at 37 ° C. for 16 hours. About the culture supernatant, ELISA [Proc.Natl.Aca
d.Sci.USA, 80,697-701, (1983)], and the productivity of protein A was examined. As a result, B. subitilis PSL1 strain of Bacillus subtilis containing plasmid pMD200 was obtained.

The construction diagram of the plasmid pMD200 is shown in FIG.

(2) Chemical synthesis of VIP-Gly gene A DNA sequence coding for VIP-Gly consisting of 29 amino acid residues, and a cleavage site for the restriction enzyme Pst I and Phe-Arg which is a kallikrein recognition sequence at its N-terminus. The DNA sequence of the spacer region that contains it, and the termination codon TAA TAG at the C-terminal side
And 122 bp VIP- with a restriction enzyme BamHI cleavage site added
The Gly (hereinafter referred to as VIP-G1) gene was chemically synthesized by a DNA synthesizer (Applied Biosystems model 381A).

The base sequence of VIP-G1 thus obtained and the corresponding amino acid sequence are shown below.

(3) Preparation of host Bacillus subtilis SPL14 strain with low protease productivity Bacillus subtilis ATCC39096 strain which is Bacillus subtilis having spoOAΔ677 gene and lincomycin resistance gene (hereinafter, referred to as Lin ^r ) is shown in Table 1. Using LB medium, shaking culture was performed at a temperature of 37 ° C. until a logarithmic growth phase.

The cells contained in 50 ml of the culture solution were collected and the method of Saito and Miura (H. Saito, K. Miura, Biochim. Biophis. Acta, 72, 619 (196
Chromosomal DNA was extracted and purified by 3)).

Bacillus subtilis DY was obtained by the competent cell transformation method using the obtained chromosomal DNA.
-16 strain was transformed. Next, the obtained transformant strain,
The culture was performed using an LB agar plate medium containing 5 μg / ml lincomycin, and a Lin ^r- introduced strain was selected.

As a result, about 800 strains of lincomycin-resistant transformants were obtained. Next, 51 sporulation-deficient strains were isolated from these lincomycin-resistant transformants as follows.

Bacillus subtilis transformed into a minimum agar medium containing 50 mg / histidine was sown and cultured at 37 ° C. for 48 hours. Strains matching the following indicators 1 to 3 were selected as sporulation-deficient strains.

1. The transformant was inoculated into a sporulation medium and the colony lost the ability to produce melanin pigment, 2. It did not show resistance to heat treatment at 80 ° C for 10 minutes, and 3. Spore formation by microscopic examination. Do not accept.

Among the obtained 173 sporulation-deficient strains, the strain with the lowest protease activity was selected as follows, and the protease activity was measured.

An LB agar plate containing 1% by weight of casein was added to a transformant deficient in preventive ability and the parent strain Bacillus subtilis.
The DY-16 strain was inoculated separately and cultured at a temperature of 37 ° C for 24 hours. Since casein is decomposed by the protease secreted by the bacterial body, the size of the halo around the bacterial body formed by the decomposition of casein is used as an index, and the protease producing ability is lower than that of Bacillus subtilis DY-16 strain. To
40 strains were separated.

Bacillus subtilis DY-16 strain and 40 strains of transformants with reduced protease activity were respectively cultured overnight in 10 ml of LB medium, and then 1 ml of the culture solution was centrifuged and present in the supernatant. The activity of protease was measured. That is, 0.2% FITC-casein (manufactured by Sigma), 100 mM Tris-hydrochloric acid buffer (pH 7.5), 2 mM calcium chloride-containing substrate solution 100 µ, protease-containing supernatant test solution 100 µ
Was added and reacted at 37 ° C. for 3 hours. After the reaction was completed, 200μ of 7.5% trichloroacetic acid was added to the reaction solution to stop the reaction, and the supernatant was obtained by centrifugation, neutralized with 500mM Tris-HCl buffer (pH8.5), and the excitation wavelength was 490nm. , Emission wavelength
Fluorescence at 525 nm was measured.

The enzyme activity for liberating 1 μM of Tyr-equivalent trichloroacetic acid-soluble peptide from casein per minute was set to 1 U and converted based on pronase (Kaken Pharmaceutical Co., Ltd.).

As a result, 4 strains were obtained in which the protease activity was extremely reduced as compared with the parent strain Bacillus subtilis DY-16 strain, and these strains were designated Bacillus subtilis SPL9 strain and SP strain, respectively.
L11 strain, SPL14 strain, and SPL39 strain.

Then, the Bacillus subtilis DY-16 strain and the transformants with reduced protease activity were treated with 10 ml of LB medium.
After culturing for 18 hours, 50 ml of Medium A medium (Journal Of B
Acteriology, 165, 796-804, 1986) and transfer the cells to culture 2
After culturing for 4 hours and 48 hours, the activity of protease in the culture supernatant was measured in the same manner as above.

The results are shown in Table 2.

From Table 2, it was confirmed that the four transformant strains had protease activity lower than that of the parent strain by 1/20 or less. In particular, it was confirmed that the SPL14 strain had the protease activity suppressed to about 3% of the parent strain DY-16. This SPL14 strain has the above-mentioned gene marker. Moreover, all of these strains have a requirement for histidine and leucine, and a plurality of auxotrophy required for the host was confirmed in a gene recombination experiment.

The protease activity of the Bacillus subtilis DY-16 strain used for transformation was compared with that of the Bacillus subtilis 207-25 strain, which is a wild strain of protease.
The results shown in are obtained. The protease activity was measured in the same manner as above by culturing the strain for 24 hours.

From Table 3, the Bacillus subtilis DY-16 strain is the wild strain 207.
Only about 1% of the -25 strains produce protease.

Bacillus subtilis SPL14 obtained as described above
The strain was used as a host for the fusion protein expression plasmid pMD235 described below.

(4) Construction of protein A-VIP-Gly fusion protein expression plasmid pMD235 The chemically synthesized VIP-G1 gene (5 μg) was phosphorylated at the 5 ′ end, and then T ₄ DNA ligase (200 units) was used to express the above formula. A plasmid containing a portion encoding the 440th Asp in protein A (mature type) represented by [II]
It was ligated to the Pst I / BamH I long chain fragment (1 μg) of pMD200, and the Bacillus subtilis Bacillus subtilis SPL14 strain was transformed by the protoplast method. From the obtained Km ^r strain, plasmid pMD235
Got When the DNA inserted into pMD235 was sequenced by the M13 dideoxy method, the VIP-G1 gene was inserted as desired.

The construction diagram of plasmid pMD235 is shown in FIG.

(5) Quantification of VIP-Gly by enzyme immunoassay Quantification of VIP-Gly by enzyme immunoassay was carried out by 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
VIP-Gly was used, which was subjected to solid-phase synthesis by a peptide synthesizer manufactured by I) and purified by reverse-phase high performance liquid chromatography to confirm the amino acid composition.

(6) Secretion expression of protein A-VIP-Gly fusion protein Bacillus subtilis Bacillus subtilis SPL14 / pMD235 strain transformed with plasmid pMD235 was used in a 500 ml Erlenmeyer flask with folds, 3.3% tryptone, 2.0% yeast. extract,
2.0% casamino acid, 1.0% glucose, 0.74% NaC
l, 0.8% Na ₂ HPO ₄ , 0.4% KH ₂ PO ₄ , 36 mM NaOH, 0.06
100 ml of medium consisting of mM MnCl ₂ (kanamycin Km 10 μg / ml
It was cultured at 37 ° C for 12 hours. After completion of the culture, phenylmethylsulfonyl fluoride (PMSF), which is a protease inhibitor, and EDTA were added to each to 10 mM, and the mixture was centrifuged to obtain a culture supernatant. Then, the secreted and expressed fusion protein was quantified by the enzyme immunoassay using the obtained culture supernatant, and 49.9 μg of the culture medium per 1
It secreted and expressed VIP-Gly.

Example 2 Secretion expression of protein A-VIP-Gly fusion protein by Bacillus subtilis-2 (1) Chemical synthesis of VIP-Gly gene DNA sequence coding for VIP-Gly consisting of 29 amino acid residues and its N-terminal restriction The DNA sequence of the cleavage region of the enzyme Hind III and the spacer region containing Phe-Arg, which is the kallikrein recognition sequence, and the termination codon TAA TAG at the C-terminal side.
And 122 bp VIP- with a restriction enzyme BamHI cleavage site added
Gly (hereinafter referred to as VIP-G2) gene was chemically synthesized by a DNA synthesizer (Applied Biosystems model 381A).

The base sequence of VIP-G2 thus obtained and the corresponding amino acid sequence are shown below.

(2) Preparation of protein A-VIP-Gly fusion protein expression plasmid pMD245 The 5'end of the chemically synthesized VIP-G2 gene (5 μg) was phosphorylated, and then T ₄ DNA ligase (200 units) was used to express the above formula. A plasmid containing up to the 187th Leu-encoding portion in protein A (mature type) represented by [II]
ligated with Hind III / BamH I long chain fragment (1 μg) of pMD20,
Bacillus subtilis Bacillus subtilis SPL14 strain was transformed by the protoplast method. From the obtained Km ^r strain, plasmid pMD24
Got 5. When the DNA inserted into pMD245 was subjected to DNA sequencing by the M13 dideoxy method, the VIP-G2 gene was inserted as desired. Plasmid pMD
The construction diagram of 245 is shown in FIG.

(3) Secretion expression of protein A-VIP-Gly fusion protein Bacillus subtilis Bacillus subtilis SPL14 / pMD245 strain transformed with plasmid pMD245 was used in a 500 ml Erlenmeyer flask with folds, 3.3% tryptone, 2.0% yeast. extract,
2.0% casamino acid, 1.0% glucose, 0.74% NaC
l, 0.8% Na ₂ HPO ₄ , 0.4% KH ₂ PO ₄ , 36 mM NaOH, 0.06
100 ml of medium consisting of mM MnCl ₂ (kanamycin Km 10 μg / ml
It was cultured at 37 ° C for 12 hours. After completion of the culture, phenylmethylsulfonyl fluoride (PMSF), which is a protease inhibitor, and EDTA were added to each to 10 mM, and the mixture was centrifuged to obtain a culture supernatant. Then, the secreted and expressed fusion protein was quantified by the enzyme immunoassay using the obtained culture supernatant, and 4.5 μg of V
It secreted and expressed IP-Gly.

[Brief description of drawings]

FIG. 1 is a construction diagram of plasmid pMD200, and FIG. 2 is a construction diagram of plasmid pMD235 and plasmid pMD245.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 125) (C12P 21/02 C12R 1: 125)

Claims (6)

[Claims] 1. A promoter or a ribosome binding site involved in gene expression, a region encoding a secretory signal and a carrier protein, and a gene encoding a vasoactive intestinal polypeptide precursor are chemically or enzymatically synthesized. DNA fragment linked via a cleavable spacer sequence is a transformant obtained by transforming a host microorganism with a fusion protein secretion expression plasmid bound to vector DNA, wherein the host microorganism is Bacillus subtilis, which lacks the ability to produce alkaline and neutral proteases and has a protease activity of less than 3% of the wild-type strain, sp.
A transformant that is a host microorganism into which oOAΔ677 mutant gene has been introduced. 2. The carrier protein is a region having an IgG-binding ability in protein A of Staphylococcus aureus.
The transformant described. 3. A Staphylococcus aureus protein A, wherein the promoter, the ribosome binding site, the secretion signal and the region encoding the carrier protein involved in gene expression are
The transformant according to claim 1, which is derived from a gene. 4. The transformant according to claim 1, wherein the vasoactive intestinal polypeptide precursor has an amino acid sequence represented by the following formula [I]. 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-Gly-
X [I] (In the formula, X is OH, Lys-OH, Arg-OH, Lys-Arg-OH,
Alternatively, it represents Arg-Lys-OH. ) 5. The host microorganism is Bacillus subtilis 104H.
The Bacillus subtilis SP011 strain (Microtechnology Research Institute No. 10987) in which the spoOA △ 677 mutant gene was introduced into the L strain, and the Bacillus subtilis SPL14 strain (in which the spoOA △ 677 mutant gene was introduced into the Bacillus dibutylis DY-16 strain The transformant according to claim 1, which is Koken Bacteria No. 10988). 6. A fusion protein obtained by culturing the transformant according to claim 1 and secreting and recovering a fusion protein in which a carrier protein and a vasoactive intestinal polypeptide precursor are linked via a spacer sequence. Manufacturing method.