JPH0751072A - Production of human growth hormone - Google Patents

Abstract

PURPOSE:To provide a microbial host-vector system suitable for the production of human growth hormone(hGH) and a process for producing hGH using the system. CONSTITUTION:The hGH-expression plasmid to be used in this production process is a recombinant DNA produced by bonding a hGH-coding DNA to the 3'-terminal of a DNA containing a promoter region originated from Bacillus brevis and the host is a mutant Bacillus brevis essentially free from protease activity on hGH. hGH can be produced in high efficiency by culturing a microorganism prepared by the transformation of the above host with the above hGH-expression plasmid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing hGH by Bacillus brevis carrying DNA encoding human growth hormone (hereinafter sometimes abbreviated as "hGH") as a heterologous gene, And said DNA and Bacillus brevis carrying said DNA.

[0002]

2. Description of the Related Art hGH consists of 191 amino acids,
Its amino acid sequence has already been revealed (Bancrof
t, FC, Expl.Cell Res ., 79 , 275-278 (1973) or Dav
id V. Goeddel et al., Nature , 281 , 544-548 (1979)). This substance has been used for a long time as a therapeutic drug for dwarfism and the like because it has growth promotion, fat metabolism, glucose metabolism and the like, and in recent years, it has been attracting attention as a therapeutic drug for senile dementia.

To date, most recombinant DNA research has been carried out using E. coli , and many heterologous proteins have already been produced in E. coli. However, in this method, since the produced heterologous protein accumulates in the bacterial cells,
It takes a lot of time and labor to extract the target product from the bacterial cells and to purify it from the extract, and it is not easy to obtain the target substance in a pure form completely.

On the other hand, microorganisms belonging to the genus Bacillus have been industrially used as producers of various extracellular enzymes since ancient times. Among these extracellular enzymes, the α-amylase gene of Bacillus amyloliquefaciens (I. Palva et al., Gene , 22 , 229 (198
3)), Bacillus lichen
iformis ) penicillinase gene (S. Chang et al., Mole
cular Cloning and Gene Regulation in Bacilli , Acade
mic Press, 659, (1982) ) and Bacillus subtilis (Bacillussubtilis)
Α-amylase gene of H. Yamazaki et al., J. Bacterio
l ., 156 , 327 (1983)) have already been cloned, and the secretory production of heterologous proteins using these promoters or signal peptides has been reported.

Bacillus subtilis is mainly used as a host for the secretory production of the above-mentioned heterologous protein in Bacillus, but since this microorganism produces a large amount of extracellular protease, it is secreted by recombinant DNA technology. The produced heterologous protein is subject to degradation, and its accumulated amount tends to be significantly reduced.

On the other hand, Utaka et al., Bacillus brevis 47, which produces almost no protease outside the cells.
(JP-A Nos. 60-58074 and 62-201583; FERM P-7224) as a host, and MWP (Middle W) which is one of the major extracellular proteins of this strain.
all Protein) (H. Yamagata et al., J. Bacteriol ., 169 , 123
9 (1987), Norihiro Tsukakoshi, Journal of Japanese Society of Agricultural Chemistry, 61, 68 (1987)
Α-amylase (Japanese Unexamined Patent Publication No. 62-201583, H. Yamagata et al., J. Bacteriol ., 169 , 1239 (1987)) and porcine pepsinogen (cormorant high). Shigezo, Proceedings of the 62nd Annual Meeting of the Japanese Society of Agricultural Chemistry p837-p838; Norihiro Tsukakoshi, Journal of the Japanese Society of Agricultural Chemistry, 61, 68 (1987)) has been successfully produced.

[0007] Cormorants also isolated Bacillus brevis HPD31 (FERM BP-1087), a strain that does not produce extracellular protease of Bacillus brevis.
Using this as a host, thermostable α-amylase (JP-A-63-
56277, Proceedings of the 62nd Annual Meeting of the Japanese Society of Agricultural Chemistry, p27, Human EGF (H. Yamagata et al., Pro.
c.Natl.Acad.Sci.USA , 86 , 3589 (1989), Japanese Patent Laid- Open No. 2-3
1682) has been successfully produced.

For hGH, E. coli (David V. Goed
del et al., Nature , 281 , 544-548 (1979), Chang C N. et al.
Gene , 55 , 189-196 (1987)), Bacillus subtilis (M. Honjo et al., J. Biot
ech., 6, 191-204 (1987 )), yeast (R.Hiramatsu et al., App
L. Environ. Microbiol. , 57 , 2052-2056 (1991)) and animal cells (Lupker J H. et al., Gene , 24 , 281-287 (1983)) have been reported as the host.

[0009]

[Problems to be Solved by the Invention]
Although production of hGH has been attempted using yeast and animal cells as hosts, it is difficult to say that all of them are satisfactory in terms of complexity of purification and productivity. Therefore, it is an object of the present invention to provide a host that enables efficient production of hGH.
It is to provide vector systems as well as to provide an efficient method for producing hGH using them.

[0010]

[Means for Solving the Problem] Utaka et al. Have developed a host-vector system for producing a heterologous protein, and found that Bacillus brevis can be advantageously used as a host, and Utilization for the production of heterologous proteins has been proposed (for example, the above-mentioned JP-A-63-56277). On the other hand, DN encoding hGH in these host strains
As a result of repeated research to create a recombinant plasmid capable of efficiently expressing A, when a promoter derived from Bacillus brevis as a host bacterium or a DNA required for secretion is used in a specific mode, hGH is also expressed. It was found to be effective.

[0011] Therefore, the above-mentioned problem is solved by recombinant DN having hGH-encoding DNA bound to the 3'end of a DNA containing a Bacillus brevis-derived promoter region.
Solved by the use of A. That is, according to the present invention, hGH is characterized in that hGH is collected from a culture by culturing the recombinant DNA, Bacillus brevis carrying the recombinant DNA and the Bacillus brevis in a nutrient medium. A method of manufacturing the same is provided.

[0012]

Specific Embodiments In addition to DNA directly isolated from human pituitary gland, DNA encoding hGH used in the present invention includes DNA chemically synthesized based on the known amino acid sequence of hGH (Ikehara et al., Proc. .Natl.Acad.Sci.USA , 8
1, may be either 5956 of (1984)). As the promoter, a promoter derived from any strain belonging to B. brevis can be used as long as it functions in Bacillus brevis (hereinafter sometimes abbreviated as "B. brevis").

For example, specifically, B. Brevis 47 (FERM P-7224) or Brevis HP
The promoter of the major extracellular protein gene of D31 (FERM BP-1087) can be mentioned. In addition to the above promoters, DNA containing a promoter region is SD
It is necessary to have a sequence, a translation initiation codon, etc., and it may further contain a part of the major extracellular protein gene.

DN containing these promoter regions
In order to bind the DNA encoding hGH to the 3'end of A, the DNA excised from the chromosome of B. brevis
(T. Adachi et al., J. Bacteriol ., 171 , 1010-1016 (198
A DNA encoding hGH may be bound to the 3'end of 9). hGH is B. Although it may be accumulated inside or outside the cells of Brevis, when hGH is accumulated outside the cells, the region containing the signal peptide is also included at the 3'end side of the DNA containing the promoter region. Need to be Examples of the signal peptide include a signal peptide of a major extracellular protein of B. brevis 47 or B. brevis HPD31. In particular,
An example is the signal peptide of MWP (Middle Wall Protein) of B. brevis 47.

An expression vector containing the promoter region and used for expressing the hGH gene is, for example, pH.
Y500, pNU200 (Japanese Unexamined Patent Publication No. 2-31682, Kozo Shigezo, Journal of Japanese Society of Agricultural Chemistry, 61, 669 (1987))
And so on. These expression vectors and the above DNA
A method for constructing an hGH expression plasmid using the method described in, for example, Molecular Cloning 2nd. Ed., A Laboratory Manual
As described in (Cold Spring Harbor Laboratory (1990)), a recombinant DNA (hGH expression plasmid) of interest can be constructed according to these methods known in the art.

The preferred hGH expression plasmid is pNU as prepared in Examples described later.
200-GH etc. can be mentioned. The host used for constructing the plasmid may be any of E. coli, Bacillus subtilis, and B. brevis microorganisms. For example, E. coli HB101, E. coli JM109, B. subtilis RM141.
( J. Bacteriol ., 158 , 1054 (1984)), B. Brevis 47
(FERM P-7224) and the like.

The host used for gene expression is
B. Brevis 47 (FERM P-7224), B. Brevis 47-5 (FERM BP-1664), B.
HGH produced by expression of the above plasmids including Brevis HPD31 (FERM BP-1087)
Any of them can be used as long as the host itself does not have a significant adverse effect. Particularly preferred are:
B. B. obtained by mutation treatment from Brevis HPD31
Brevis 31-OK (FERM BP-4573) can be mentioned. Since this mutant has substantially no protease activity on hGH, the culture (especially,
HGH produced and accumulated in (extracellular) can be stably maintained.

To transform host B. brevis with the above-mentioned hGH expression plasmid, for example, the method of Takahashi et al. ( J. Bacteriol ., 156 , 1130 (1983)) or the method by electroporation (H. Takagi et al., Agric.Biol.Che
m ., 53 , 3099-3100 (1989)) and the like. The transformants thus created produce a large amount of hGH by culturing them in a nutrient medium, and most of them are secreted outside the cells. In addition, when B. brevis 31-OK is used as a host, hGH secreted to the outside of the cells can be stably maintained.
Can be efficiently collected.

The nutrient medium used for culturing contains a carbon source, a nitrogen source and, if necessary, inorganic salts. Alternatively, the culture may be carried out using a synthetic medium mainly containing sugar and inorganic salts.
When using a auxotrophic strain, it is desirable to add nutrients required for its growth to the medium. Further, if necessary, antibiotics, antifoaming agents and the like may be added to the medium. As the culture condition, the initial pH of the medium is 5.0 to
It is adjusted to 9.0, preferably 6.5 to 7.5. The culture temperature is usually 15 ° C to 42 ° C, preferably 24 to 37 ° C, and the culture time is generally 16 to 360 hours, preferably 2
4 to 144 hours.

After the completion of the culture, in order to collect hGH from the culture, the cells and the supernatant are separated by, for example, centrifugation or filtration. The hGH produced in the microbial cells is extracted by crushing the microbial cells by a conventional method in the technical field, for example, ultrasonic crushing method, French press method, etc., and adding a surfactant if necessary. Then, hGH contained in the culture supernatant or bacterial cell extract thus obtained is
The target hGH can be obtained by purification by a commonly used protein purification method, for example, salting out, isoelectric precipitation, gel filtration, ion exchange, various chromatographies such as reverse phase, and the like.

The obtained hGH can be quantified by an enzyme immunoassay using an anti-hGH antibody or HPLC. In addition, hGH can be quantified also from the adipocyte differentiation activity against 3T3-L1 cells (cell culture technology (second edition) edited by the Japan Tissue Culture Society).

[0022]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 . Of human growth hormone (hGH) expression vector
Construction and transformation of the adjustment made plasmid pGH-L9 (Ikehara et al, Proc.
Sci . USA ., 81 , 5956-5960 (1984)), a 565 bp HinfI-SalI fragment encoding hGH was isolated. A DNA linker was chemically synthesized in order to connect the signal peptide of MWP and hGH. The synthetic linker and the HinfI-SalI fragment containing the hGH gene were ligated with T4 DNA ligase to give 596 bp N
A coI-SalI fragment was obtained.

On the other hand, Bacillus brevis 47 (FERM
One of the major membrane proteins of P-7224) MWP (Middle
The plasmid pBR-AN2 containing the C-terminal region among the signal peptides of Wall Protein (JP-A-2-25787)
No. 6), the 27 bp NcoI-SalI fragment was removed and the above 596 bp NcoI-SalI fragment was removed.
The fragment was inserted to obtain the plasmid pBR-AN2-GH.

The plasmid pBR-AN2-GH was treated with restriction enzymes ApaLI and HindIII to give 656 bp.
The ApaLI-HindIII fragment was isolated. On the other hand, the plasmid pNU200 (see Ushio Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 61, 669-676 (1987)) was used as a restriction enzyme ApaLI, H.
cleaved with indIII, the large fragment and 656 bp above

ApaLI-HindIII fragment and T4D
Using the reaction solution ligated with NA ligase, Takahashi
( See J. Bacteriol ., 156 , 1130 (1983)), Bacillus brevis 47-5 (FERM BP-1
664, IFO 14698). The resulting erythromycin-resistant transformants were subjected to an alkaline extraction method (Molecular Cloning 2nd ed., A laboratory Ma
A plasmid was isolated by Nual, Cold Spring Harbor Laboratory (1989)) and named pNU200-GH.

Further, the plasmid pNU200-GH was digested with the restriction enzymes ApaLI and BclI to obtain an ApaLI-BclI fragment containing the hGH gene. Also, MWP
Plasmid pNUR2L4 having an improved signal sequence R2L4 (Fig. 5 (R2L4); SEQ ID NO: 2) in which 3 Leu were added to the hydrophobic region of the signal sequence (Fig. 5 (MWP); SEQ ID NO: 1) and 2 Arg were added to the positively charged region. Was digested with restriction enzymes ApaLI and BclI to obtain about 3.8 kb.
The pApaLI-BclI fragment and the above-mentioned DNA fragment containing the hGH gene were ligated with T4 DNA ligase to obtain a plasmid pNUR2L4-GH.

The above plasmids pNU200-GH and pNUR2L4-GH were prepared by the method of Takahashi ( J. Bacteri.
ol., 156, by 1130 (1983)) was introduced into Bacillus brevis 31-OK (FERM BP-4573 ), to obtain a transformant. A stable strain was isolated from the obtained transformants, and this was transformed into Bacillus brevis 31-OK (pNU
200-GH) and Bacillus brevis 31-OK (p
NUR2L4-GH).

Example 2 Cultivation of transformants and hGH
Secretory production of Bacillus brevis 31 obtained in Example 1
-OK (pNU200-GH), Bacillus brevis 3
1-OK (pNUR2L4-GH) and its control Bacillus brevis 31-OK (pNU200) and Bacillus brevis 31-OK (pNUR2L4) were polypeptone 3%, yeast extract 0.2%, glucose 3%, M
_{gSO 4 · 7H 2 O 0.01%} , CaCl 2 · 7H 2
O 0.01%, MnSO ₄ .4H ₂ O 0.001
_{%, FeSO 4 · 7H 2 O} 0.001%, ZnSO 4
7H ₂ O 0.0001% and erythromycin 1
The cells were cultured in a medium containing 0 mg / L (pH 7.2) at 30 ° C for 3 to 6 days.

The above culture broth was centrifuged and the supernatant hG
The H concentration was determined by an enzyme immunoassay using an anti-hGH antibody (Table 1). In addition, as a standard product of hGH, Biopr
The product purchased from oducts (Seikagaku Corporation) was used.

[0030]

[Table 1]

Next, the above-mentioned culture supernatant was subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (see Nature , 227 , 680 (1970)), and then Burnette's method ( Anal . Biochem ., 112 , 195) . (See (1981))
Western blotting was performed using the GH antibody. As a result, Bacillus brevis 31-OK (pNU200
-GH) and Bacillus brevis 31-OK (pNU
HGH was observed in the culture supernatant of R2L4-GH), and its molecular weight was in agreement with that of the standard product (FIG. 1).

Example 3 Purification of hGH 100 ml of the culture supernatant obtained in Example 2 was subjected to ultrafiltration (Fitr
on, Minisette Omega, 10k cutoff)
Ion exchange (Pharmacia), Inverse layer (Pharmacia), Inverse layer (Wa
ters) in the order of chromatography to obtain 2.3 mg of a purified hGH preparation.

Example 4 Properties of purified hGH preparation The various properties of the purified hGH preparation obtained in Example 3 were as follows. Retention time of HPLC As a result of examination by reverse phase HPLC, the obtained purified hGH preparation was almost single (Fig. 2), and its retention time was hGH standard preparation.
It coincided with that of (Bioproducts). As a result of examination by 16% polyacrylamide gel electrophoresis containing PAGE 0.1% SDS, the purified preparation showed the same behavior as the standard preparation under both reducing and non-reducing conditions (Fig. 3). .

N-Terminal Amino Acid Sequence Pulse Liquid Automated Edman Degradation (Applied Biosystems
As a result of investigating the N-terminal amino acid sequence of the purified standard using the product (473A type), the sequence of the purified standard was found in the literature (Bancroft, F.
C., Expl . Cell Res ., 79 , 275-278 (1973), David V. Goedde
l et al., Nature , 281 , 544-548 (1979)).

[0035]

[Table 2]

Biological activity As a result of examining the biological activity of hGH using the ability of 3T3-L1 cells to differentiate into adipocytes as an index (see Tissue Culture Technology (Second Edition) p110-115, edited by the Japan Tissue Culture Society), purification was performed. The standard product had the same biological activity as the standard product (Fig. 4).

Reference Example 1 Bacillus Brevis
Brevis) 31-OK Creation The plasmid pNU200-GH obtained in Example 1 was transformed with Taka
The parent strain Bacillus brevis HPD3 by the method of hashi et al.
1 (JP-A-63-56277, FERMBP-1
087), Bacillus brevis HPD31 (p
NU200-GH) was obtained. The transformant was diluted and suspended in a T2 medium, and smeared on a T2 plate, followed by ultraviolet rays (2
J / m ² ).

After culturing at 30 ° C. overnight, the resulting colonies were inoculated into 5YC medium and cultured at 30 ° C. for 6 days. The culture broths collected on the 3rd and 6th days were collected and
The amount of GH was measured by enzyme immunoassay. At this time, 3
One strain in which the amount of hGH in the culture supernatant increased from the day to the sixth day was selectively isolated. When the mutant strain and the parent strain are compared, the amount of hGH in the culture supernatant of the parent strain decreases from the 3rd day to the 6th day, whereas the obtained mutant strain increases the hGH amount even after the 3rd day. It showed a remarkable difference. In order to remove the plasmid from the mutant strain, the mutant strain was repeatedly subcultured in T2 medium containing no erythromycin, and a strain that was sensitive to erythromycin and did not produce hGH was easily obtained. This strain was named Bacillus brevis 31-OK.

Bacillus brevis 31-OK (FERM
The mycological properties of BP-4573) are:
It exhibited almost the same properties as Brevis HPD31 (see Japanese Patent Laid-Open No. 63-56277) except that the action on a specific heterologous protein was different. The main properties are shown below.

[0040]

[0041]

Decomposition of gelatin-Production of acid from glucose-Xylose decomposition-Lactose decomposition-Maltose decomposition-Arginine hydrolysis-Viable pH 5.5-8.5

Morphological properties Cell size Liquid medium; 0.9-1.2x
3.9 to 5.8 μm agar medium; 0.9 to 1.2 × 2.9 to 4.2 μm cell shape bacillus cell polymorphism presence / absence motility presence / absence

Reference Example 2 Preparation of plasmid pNUR2L4
Ltd. (1) restriction enzyme EcoRI to construct pNU200 plasmid M13MWP, was digested with HindIII (Takara Shuzo Co., Ltd.), the obtained DNA fragment of approximately 670 bp, of plasmid M13mp18 EcoRI, Hind
Inserted into the digested with III, plasmid M13MWP
Got The nucleotide sequence from the MWP promoter to the signal sequence is shown in Fig. 5 (MWP) (SEQ ID NO: 1).

(2) Construction of plasmid M13R2 Using the M13MWP single-stranded DNA obtained above as a template, synthetic primer R2 (FIG. 6) (SEQ ID NO: 3) was used for site-directed mutagenesis. Site-directed mutagenesis is performed using the Amersham kit (Oligonucleotide-directed in vitro mutagene
sis system, Amersham). Next, the nucleotide sequence of the DNA was confirmed, and the obtained target plasmid was designated as M13R2.

(3) Construction of plasmid M13L4 Using the plasmid M13MWP single-stranded DNA as a template and synthetic primer L4 (FIG. 6) (SEQ ID NO: 4), a plasmid was prepared in the same manner as in Reference Example 2- (2). M13L4
Got (4) Construction of plasmid pNUR2L4 M13R2 obtained in (2) was digested with EcoRI and HpaI, and the obtained 228 bp DNA fragment was obtained in (3).
1 obtained by digesting 13L4 with HpaI and HindIII
08bp DNA fragment, pNU200 EcoR
The plasmid p was inserted into the digested product with I and HindIII.
NUR2L4 was obtained. This signal sequence (R2L4) is shown in FIG. 5 (R2L4) (SEQ ID NO: 2).

[0047]

According to the present invention, human growth hormone (h
A host-vector system capable of efficiently producing GH) is provided. That is, Bacillus brevis was used as the host, and h was added to the 3'end of the DNA containing the promoter region derived from Bacillus brevis as the hGH expression plasmid.
HGH can be efficiently produced by using a recombinant DNA in which a DNA encoding GH is bound. In particular, it is advantageous to use a mutant Bacillus brevis as a host, which exhibits substantially no protease activity against hGH.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 73 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: genomic DNA Origin organism name: Bacillus brevis Strain name: 47 (FERM P-7224) Features: Signal sequence of Bacillus brevis 47 MWP Sequence ATG AAA AAG GTC GTT AAC AGT GTA TTG GCT AGT GCA CTC GCA CTT 45 Met Lys Lys Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu 1 5 10 15 ACT GTT GCT CCC ATG GCT TTC GCT GCAG 73 Thr Val Ala Pro Met Ala Phe Ala 20

SEQ ID NO: 2 Sequence length: 88 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Synthetic DNA Origin plasmid pNUR2L4 Characteristics: Signal sequence of Bacillus brevis 47 MWP Improved signal sequence ATG AAA AAA AGA AGG GTC GTT AAC AGT GTA TTG CTT CTG CTA GCT 45 Met Lys Lys Arg Arg Val Val Asn Ser Val Leu Leu Leu Leu Ala 1 5 10 15 AGT GCA CTC GCA CTT ACT GTT GCT CCC ATG GCT TTC GCT GCAG 88 Ser Ala Leu Ala Leu Thr Val Ala Pro Met Ala Phe Ala 20 25

SEQ ID NO: 3 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CACTGTTAAC GACCCTTCTT TTTTTCATGA CCTTGTGTTC 40

SEQ ID NO: 4 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CCGTGCACTA GCTAGCAGAA GCAATACACT GTTAACG 37

[Brief description of drawings]

FIG. 1 shows the results of Western blotting after subjecting hGH and standard hGH in the culture supernatant obtained in Example 2 to SDS-polyacrylamide gel electrophoresis.

FIG. 2 is a reverse layer HP of the hGH purified sample obtained in Example 4.
The result of LC is shown. (The horizontal axis is the holding time (minutes), ↓ is h
The time (minutes) when GH was eluted is shown. )

FIG. 3 SDS- of the purified hGH preparation obtained in Example 4
The results of comparing the polyacrylamide gel electrophoresis patterns with those of the standard product under both reducing and non-reducing conditions are shown.

FIG. 4 is a graph showing a comparison of the biological activity of the purified hGH preparation obtained in Example 4 and the standard preparation with respect to the 3T3-L1 cell differentiation potential as an index.

FIG. 5: Bacillus brevis 47 MWP signal sequence (shown as MWP) (SEQ ID NO: 1) and a modified signal sequence of this MWP signal sequence (R2L
4) (shown as 4) (SEQ ID NO: 2).

FIG. 6 is a diagrammatic representation of synthetic primers (SEQ ID NOs: 3 and 4) used for site-directed mutagenesis of DNA encoding the signal sequence of MWP.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:08) (C12P 21/02 C12R 1:08) (72) Inventor Tsutomu Kajino Aichi Aichi 1 in 41 Chuo, Yokota, Nagakute-machi, Gunma District Toyota Central Research Institute Co., Ltd. (72) Inventor Yoko Ota 1 in 41, Yokota, Nagakute-machi, Aichi-gun, Akita Prefecture (72) Invention Person Hirai Masana 41, Nagachote-cho, Nagakute-cho, Aichi-gun, Aichi Prefecture 1st in Yokota Central Research Institute Co., Ltd. (72) Inventor, Yukio Yamada 1-41, Nagakute-cho, Aichi-gun, Aichi-gun 1st place, Yokota, Toyota Chuo In the laboratory (72) Inventor Fumihiko Hoshino 1 at 41, Yokomichi, Nagakute-cho, Aichi-gun, Aichi-gun Toyota Central Research Institute Co., Ltd.

Claims (6)

[Claims] 1. Bacillus brevi
s ) A recombinant DNA obtained by ligating a DNA encoding human growth hormone to the 3'end of a DNA containing a promoter region derived from s ). 2. A MWP (Middle) of Bacillus brevis 47 is added to the 3'-end side of a DNA containing a promoter region.
Recombinant DNA according to claim 1, containing a signal sequence of Wall Protein). 3. The signal sequence is an improved signal sequence R2.
The recombinant DN according to claim 2, which is L4 (SEQ ID NO: 2).
A. 4. The recombinant DN according to claim 1, 2, or 3.
Bacillus brevis holding A. 5. A method for producing human growth hormone, which comprises culturing Bacillus brevis according to claim 4 in a nutrient medium and collecting human growth hormone from the culture. 6. The method for producing human growth hormone according to claim 5, wherein the Bacillus brevis is Bacillus brevis 31-OK (FERM BP-4573).