JP3164181B2 - Novel expression vector, microorganism carrying the expression vector, and method for producing useful substance using the microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to biotechnology, and more particularly, the present invention relates to a novel expression vector, a microorganism having a vector obtained by binding a foreign gene to the expression vector, and a microorganism comprising the same. The present invention relates to a method for producing a gene product, which comprises culturing a microorganism, producing and accumulating a foreign gene product in a culture, and collecting the gene product.

[0002]

2. Description of the Related Art Udaka et al., Bacillus brevis (Baci).
lrus brevis) does not produce proteases.
And found that there were many strains of Bacillus subtilis.
Levis 47 (JP-A-60-58074, JP-A-62-2)
01583) major extracellular protein [H. Yamagata
a et. al. , J. et al. Bacteriol. ,16
9, 1239 (1987): Norihiro Tsukagoshi, Japanese Society of Agricultural Chemistry
magazine,6168 (1987) and JP-A-62-201.
No. 583, “outer wallpro”
tein and middle wall prot
ein "and" extracellular protein ".]
Gene promoter and one of the major extracellular proteins
MW protein (middle wall prot)
ein) using the region encoding the signal peptide
A secretory vector was prepared and α-amilla
(JP-A-62-201583, H. Yamagata
aet. al. , J. et al. Bacteriol. ,169,
1239 (1987) and porcine pepsinogen (Shigeru Udaka)
3. Japanese Society of Agricultural Chemistry 1987 Annual Meeting Abstracts, p8
37-838; Norihiro Tsukagoshi, Journal of the Japanese Society of Agricultural Chemistry,61, 6
8 (1987)).

Also, Takagi et al., A strain of Bacillus brevis that does not produce protease extracellularly, Bacillus brevis HPD31 (this strain is referred to herein as Bacillus brevis H102 (FERM BP-108).
7), and using this as a host, highly secretory production of thermostable α-amylase (Agric. Bio).
l. Chem. , 58 , 2779-2880 (198
9)) and high secretory production of human EGF by Yamagata et al.
c. Natl. Acad. Sci. USA, 86 , 35
89-3593 (1989)).

[0004]

The productivity of a foreign gene product using Bacillus brevis as a host bacterium is dramatically improved as compared with the production amount before applying genetic recombination and a system using Escherichia coli as a host bacterium. are doing. However, conventionally, in order to cause the expression of a foreign gene product, Bacillus
A brevis-replicatable vector is used, and a promoter for expressing a foreign gene product, an SD sequence, a secretory signal sequence starting from the translation initiation codon ATG, and a foreign protein gene are connected downstream thereof. In some genes, expression of a foreign gene product did not occur or was unlikely to occur even when such a conventional technique was used. It has been desired to develop a vector capable of efficiently expressing a foreign gene product whose expression does not or does not easily occur in such conventional techniques.

[0005]

Means for Solving the Problems Bacillus brevis is a host bacterium capable of highly expressing foreign genes derived from both prokaryotic and eukaryotic cells. Among them, a high expression system for hEGF has been developed in Bacillus brevis ( JP-A-4-27
8091, Japanese Patent Application No. 4-216605), it can be said that it is one of the most successfully expressed genes among foreign gene products. The present inventors have focused on this fact and as a result of research, as a result, by inserting the entire gene encoding the amino acid sequence of hEGF or a part including the N-terminus into a part of the vector, the conventional art The present inventors have succeeded in developing an expression vector capable of expressing a foreign gene product, which has been difficult to express, and completed the present invention.

The nucleotide sequence encoding the hEGF amino acid sequence (SEQ ID NO: 1) used in the present invention may be any nucleotide sequence encoding the hEGF amino acid sequence of SEQ ID NO: 1 in the sequence listing shown in Table 1 below. It may be an array. For example, a known hEGF nucleotide sequence (Proc. Nat
l. Acad. Sci. USA, 88 , 3589-35.
93 (1989)) or the entire hEGF base sequence synthesized from the hEGF amino acid sequence (Japanese Patent Application No. 4-216605) or a part including the N-terminus. it can. These are known synthetic methods (Itakura, K., JJ Rossi,
and R. B. Wallace. , Annu. Re
v. Biochem. , 53 , 323 (1984)).

[0007]

[Table 1]

In the present invention, since the target foreign gene product is obtained as a fusion protein with hEGF, a protease-recognizing amino acid is located downstream of the hEGF gene in order to cleave the target foreign gene product and hEGF by a protease in a later step. A base sequence encoding the sequence may be inserted. The nucleotide sequence encoding the recognition amino acid sequence of the protease to be inserted may be any sequence that can be easily cleaved with hEGF after obtaining the foreign gene product of interest. For example, a base sequence ST containing a base sequence encoding an amino acid sequence cleaved by thrombin (FIG. 5)
And a base sequence BF (FIG. 6) containing a base sequence encoding an amino acid sequence cleaved by Factor-Xa.

The promoter, SD and signal peptide-encoding gene may be any as long as they function in the host, but Bacillus brevis H102 (FER)
MBP-1087) -derived gene encoding promoter, SD, signal peptide (J. Bacterio)
l. , 172 , 1312-1320 (1990)). As a method for constructing a plasmid, a conventional method is appropriately used, for example, Molecular
Cloning, A Laboratory Manua
1, Cold Spring Harbor Labo
rat (1982). Expression vector pH of the present invention constructed under the above conditions
Foreign gene X linked to T.hEGF.R is insulin-like growth factor-I (I
GF-I), hepatitis C envelope protein (HCV-E)
nv) and the like. If these are inserted into an in-frame and the microorganism is transformed, it is possible to obtain a transformant that can efficiently produce a foreign gene product that has been difficult to express by conventional techniques.

The microorganism transformed with the vector incorporating the gene encoding the foreign gene product may be any microorganism capable of expressing the vector and promoter. For example, Bacillus brevis 47 (Japanese Patent Application Laid-Open No.
58074, JP-A-62-201583), Bacillus
Brevis H102 (FERM BP-1087), etc., but Bacillus brevis H102 is preferably used. The method for transforming the microorganism may be a known method, for example, the method of Takahashi et al. (J. Ba).
cterol. , 156 , 1130 (1983)) or the method of Takagi et al. (Agric. Biol. C).
hem. , 53 , 3099-3100 (1989)).

The medium used for culturing the obtained transformant may be any medium as long as the transformant can grow and produce the desired foreign gene product.

[0012] Examples of the carbon source contained in the medium include glucose, glycerol, starch, dextrin, molasses, urea, and organic acids. Examples of the nitrogen source contained in the medium include casein, polypeptone, meat extract, yeast extract, organic nitrogen sources such as casamino acid and glycine, and inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate. Moreover, you may culture | cultivate using the synthetic medium which mainly has a sugar and an inorganic nitrogen source. Bacteria showing auxotrophy may be supplemented with nutrients necessary for their growth to the medium. Examples of the nutrient include amino acids, vitamins, nucleic acids, salts and the like.

If necessary for culturing, antibiotics such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine,
Ampicillin and the like are added. If necessary, an antifoaming agent such as soybean oil, lard oil, various surfactants and the like may be added to the medium.

The initial pH of the medium is from 5.0 to 9.0, and more preferably from 6.0 to 8.0. Culture temperature is usually 1
5 ° C to 42 ° C, the culture time is 24 to 96 hours.

According to the present invention, by culturing the transformant under the above-mentioned conditions, various foreign gene products such as IGF-I and HCV-Env, which could hardly be expressed in the culture by the conventional method, can be transformed with hEGF. It is efficiently produced and accumulated as a fusion protein.

When the fusion protein is produced in the cells, known methods such as microfluidizer (manufactured by Microfluidex), ultra-long-wave treatment, French press, etc. (Basic Experimental Methods for Proteins and Enzymes, Nankodo, 1 ~ 8 pages (1
985)), the cells can be crushed and collected outside the cells. If it was produced as an enclosure,
It can be solubilized with urea, guanidine hydrochloride or the like. When produced outside the cells, the cells and the supernatant may be separated by centrifugation, membrane filtration, or the like.

The recovered fusion protein can be obtained by a conventional method, for example, membrane treatment, ammonium sulfate fractionation, chromatography, etc.
The enzyme can be purified by Basic Experimental Methods, Nankodo, (1985). Further, at this time, if affinity chromatography for hEGF is performed, the fusion protein can be purified efficiently.

Although the purified fusion protein may be used as it is, it is preferable that hEGF is removed and the fusion protein is used in the form of only the target protein. The separation of hEGF and the target protein is performed by a protease that cleaves the amino acid sequence encoded by the inserted base sequence R, such as thrombin or Facto.
The fusion protein is treated with r-Xa or the like, and the target protein and hE
Can be cut into GF. Cut hEGF into hE
The target protein can be obtained by removal by affinity chromatography for GF or the like. Further, after treating with thrombin, Factor-Xa, or the like, the target protein may be purified by the above-described conventional protein purification method.

Now, the present invention will be described in further detail with reference to Examples.

[0020]

Example 1 Expression Vector pHT / hEGF / ST / I
Construction of GF-I and production of IGF-I by transformant

(1) Plasmid pHT110 · hEGF
Construction of B. brevis HP926 (FERM BP-53
82) is digested with ApaLI and BamHI, and then treated with alkaline phosphatase (BAP treatment) to obtain 3.4 kb of DN.
A fragment was obtained. To this, synthesized by a conventional method (Itakur
a, K .; , J. et al. J. Rossi, and R.S. B. Wa
lrace. , Annu. Rev .. Biochem. ,
53 , 323 (1984)), the MWP signal peptide (H. Yamagata et. Al., J. Ba).
cterol. , 169 , 1239 (1987)).
bp synthetic DNA (FIG. 7) was ligated with T4 ligase to obtain a plasmid pHT110.hEGF (FIG. 8).

(2) Plasmid pHT / hEGF / ST
Next, plasmid pHT110 · hEGF was replaced with SphI and Bph.
The enzyme was digested with glII, then blunted at the cleavage site with T4 polymerase, and synthesized by a conventional method (Itakura, K., et al.
J. J. Rossi, and R.S. B. Wallac
e. , Annu. Rev .. Biochem. , 53 , 3
23 (1984)) and the synthetic DNA (ST) encoding the amino acid sequence recognized by thrombin shown in FIG.
Ligated using ligase, plasmid pHT-hEGF
-ST was obtained (Fig. 9).

(3) Plasmid pHT / hEGF / ST
Construction of IGF-I The gene encoding IGF-I shown in FIG. 10 was synthesized by a known synthesis method (Itakura, K., JJ Rossi,
and R. B. Wallace. , Annu. Re
v. Biochem. 53 , 323 (1984)), and inserted into the PstI-HindIII site of plasmid pUC19 (manufactured by Takara Shuzo) to insert plasmid pUC19.
IGF-I was obtained.

Next, the plasmid pIGF-I was digested with PstI, the digested portion was blunted with T4 polymerase, and then digested with HindIII to obtain a 220 bp IGF-I gene fragment.

Next, the plasmid pHT.hE
GF · ST is digested with Aor51HI and HindIII,
After the BAP treatment, the previously obtained IGF-I gene fragment was
Ligated using 4 ligases and plasmid pHT-hEGF
ST-IGF-I was obtained (FIG. 11).

(4) Secretory production of IGF-I by the transformant Next, pHT-hEGF-ST-IGF-I
Brevis HPD31 (FERM BP-1087) was transformed to obtain a transformant retaining pHT / hEGF / ST / IGF-I. pHT ・ hEGF ・ ST ・ IG
Bacillus brevis HPD31 retaining FI was added to 5 'PY medium containing erythromycin (10 µg / ml) (Agric. Biol. Chem., 53 , 227).
9, (1989)) and cultured at 30 ° C. for 1 day. 1 ml of the preculture was added to 5'PY medium 1
The cells were inoculated into 00 ml and cultured with shaking at 30 ° C. for 4 days. After the culture, the cells were collected by centrifugation, and the cells were crushed with a microfluidizer, followed by centrifugation to collect a precipitated fraction. The precipitated fraction was subjected to Tris-Triton (0.5% Trito).
nX-100, 10 mM EDTA, 20 mM Tri
s-HCl (pH 8.0)) (50 ml ×
2), Tris / Urea / DTT (8M Urea,
10 mM DTT, 20 mM Tris (pH 8.0)
Dissolved in 50 ml. The solubilized fraction containing the hEGF-IGF-I fusion protein (hEGF-ST-IGF-I) was subjected to anion-exchange chromatography (Whatman DE53,
Tris · Urea / DTT). After the adsorption, the column was sufficiently washed with Tris / Urea / DTT, and then eluted with a gradient of NaCl 0-0.5M, followed by elution with hEGF / ST / IGF-I.
Was recovered. The recovered hEGF / ST / IGF-I is T
BS (100 mM NaCl, 20 mM Tris-H
Urea was removed by dialysis against Cl (pH 8.0)).

After dialysis, the fraction containing hEGF.ST.IGF-I was applied to an affinity column to which an anti-hEGF antibody had been adsorbed to adsorb hEGF.ST.IGF-I. After washing the column, the column was washed with 4M sodium iodide (NaI).
EGF-ST-IGF-I was eluted and recovered, and purified hE
GF-ST-IGF-I was obtained.

Next, purified hEGF.ST.IGF-I was added to 2
Thrombin treatment at 3 ° C. for 8 hours, hEGF and IGF-I
These were subjected to the same hEGF affinity column as described above to adsorb hEGF, and the unadsorbed fraction (IGF
The fraction containing -I) was collected.

These were dialyzed against TBS and purified IGF-
I was obtained.

[0030]

Example 2 Purified hEGF.ST.IGF-I, Purified I
Amino acid composition and N-terminal and C-terminal amino acid sequences of GF-I Since the hEGF / ST / IGF-I obtained in Example 1 is not secreted outside the cells, the MWP signal peptide (H. Yamagata et. , J. Bact
eriol. , 169 , 1239 (1987)).
It seems that the amino acid is expressed in an added form. The primary sequence of the predicted amino acid is shown in FIG.

The purified hEGF.ST.IG obtained in Example 1
After hydrolyzing FI with 6N hydrochloric acid for 8 hours, the amino acid composition was measured by the PITC method (PICO-TAG amino acid analysis system manufactured by Waters). The measured values of the amino acid composition are shown in Table 3 below together with the amino acid composition (theoretical value) determined from the predicted primary sequence of the amino acid of hEGF.ST.IGF-I (FIG. 12). Both values were almost the same.

[0032]

[Table 3]

The N-terminal amino acid sequence of hEGF.ST.IGF-I was analyzed by Edman degradation (P. Edman, A.
rch. Biochem. Biophys. , 22 , 4
75 (1949)) is shown in Table 4 below. The result is the expected N-terminal amino acid sequence (Fig. 1
Completely matched with 2). Furthermore, hEGF ・ ST ・ IG
The C-terminal amino acid sequence of FI was subjected to a limited digestion method using carboxypeptidase Y (RP Ambler, Met).
hod in Enzymology, 25 , 143
(1972)), the results of which are shown in Table 4. This also completely coincided with the expected C-terminal amino acid sequence of hEGF.ST.IGF-I (FIG. 12). From the above results, it was confirmed that the obtained hEGF.ST.IGF-I was a target protein.

[0034]

[Table 4]

Next, the amino acid composition, N-terminal and C-terminal amino acid sequences of the purified IGF-I obtained in Example 1 were examined in the same manner as described above. The measured values of the amino acid composition are shown in Table 5 below together with the theoretical values obtained from the predicted primary sequence of the amino acid (FIG. 13). Both values were almost the same.

[0036]

[Table 5]

The N-terminal and C-terminal amino acid sequences are shown in Table 6 below. Both the N-terminal and C-terminal amino acid sequences are the predicted primary amino acid sequences (FIG. 13).
(N-terminal amino acid residues Gly, S of the sequence recognized by thrombin when cut with thrombin
er remains). From the above results, the obtained IGF
-I was confirmed to be the target protein.

[0038]

[Table 6]

[0039]

Example 3 Expression Vector pHT / hEGF / BF / H
Construction of CV-Env and HCV-Env by transformant
Production of】

(1) Plasmid pHT / hEGF / BF
Construction of pHT110 hEGF obtained in Example 1 was digested with BglII, and the cleavage site was blunted with T4 polymerase.
Synthesized by a conventional method (Itakura, K., JJ Ro)
ssi, and R.S. B. Wallace. , Ann
u. Rev .. Biochem. , 53 , 323 (198
4) The synthesized DNA (BF) encoding the amino acid sequence recognized by Factor-Xa shown in FIG. 6 was ligated using T4 ligase, and the plasmid pHT.hEGF.B was ligated.
F was obtained (FIG. 14).

(2) Plasmid pHT / hEGF / BF
-Construction of HCV-Env A known HCV-Env gene sequence (Japan JE
xp. Med. , 60 , 167, (1990)) (Itakura, K., JJ Rossi,
and R. B. Wallace. , Annu. Re
v. Biochem. , 53 , 323 (1984)) and the 577 bp HCV-Env gene (FIG. 15) (HC
The nucleotide sequence (5) obtained by removing the restriction enzyme sites added to the N-terminal and C-terminal of the V-Env gene (577 bp)
73 bp) is inserted into the PstI-EcoRI site of plasmid pSPT18 (Boehringer Mannheim) as shown in Table 2 below as SEQ ID NO: 2 in the sequence listing, and
Plasmid pHCV · E1 having CV-Env gene
I got

[0042]

[Table 2]

After digesting pHCV · E1 with PstI,
The cut portion was blunted with 4 polymerase and digested with EcoRI to obtain a 580 bp envelope gene fragment.

The previously prepared plasmid pHT · hEGF
・ Digest BF with StuI and EcoRI, and after BAP treatment,
The envelope gene fragment obtained above was ligated using T4 ligase, and the plasmid pHT / hEGF / BF / HCV was ligated.
-Env was obtained (FIG. 16).

(3) Secretory Production of HCV-Env by Transformant Next, Bacillus brevis HPD31 (FERM BP-1087) was used for pHT, hEGF, BF, HCV-Env.
Was transformed into pHT, hEGF, BF, HCV-En.
A transformant retaining v was obtained.

PHT / hEGF / BF / HCV-Env
Brevis HPD31, which is maintained in a 5 ′ PY medium (Agri), containing erythromycin (10 μ / ml).
c. Biol. Chem. , 53 , 2279 (198
9)), and cultured at 30 ° C. for 1 day. Preculture 1
ml was inoculated into 100 ml of 5'PY medium having the same composition as above, and cultured with shaking at 30 ° C for 4 days. After the culture, the cells were collected by centrifugation, and the cells were crushed with a microfluidizer, followed by centrifugation to collect a precipitated fraction. The precipitated fraction is
is-Triton (0.5% Triton X-10
0,10 mM EDTA, 20 mM Tris-HCl
(PH 8.0)) and washed (50 ml × 2 times),
s · Urea / DTT (8 M Urea. 10 mM D
TT, 50 mM Tris (pH 8.0)).

The solubilized fraction containing the fusion protein of hEGF and HCV-Env (hEGF-BF-HCV-Env) was anion-exchanged (Whatman DE53,
(Tris-Urea / DTT equilibrated)). After the adsorption, the column was sufficiently washed with Tris / Urea / DTT, and then NaCl 0-0.5
Eluted with a gradient of M, hEGF, BF, HCV
-Collected Env.

The recovered hEGF / BF / HCV-Env
Is TBS (100 mM NaCl, 20 mM Tris
-HCl (pH 8.0)) to remove Urea.

After dialysis, the fraction containing hEGF.BF.HCV-Env was applied to an affinity column to which an anti-hEGF antibody had been adsorbed to adsorb hEGF.BF.HCV-Env. After washing the column, 4M sodium iodide (Na
In (I), hEGF.BF.HCV-Env was eluted and collected to obtain purified hEGF.BF.HCV-Env.

Next, purified hEGF.BF.HCV-En
v at 23 ° C. for 8 hours with Factor-Xa
F and HCV-Env and cleave them into the same hE
HEGF is adsorbed on a GF affinity column,
Unadsorbed fractions (fractions containing HCV-Env) were collected to remove cleaved hEGF.

These were dialyzed against TBS and purified HCV-
Env was obtained.

[0052]

Example 4 Amino acid composition and N-terminal and C-terminal amino acid sequences of purified hEGF-BF-HCV-Env and purified HCV-Env-hEGF-BF-HCV- obtained in Example 3
Since Env is not secreted outside the cells, an MWP signal peptide (H. Yamagata et. Al.,
J. Bacteriol. , 169 , 1239 (198
It is considered that the amino acid is expressed in a form in which 23 amino acids of 7)) are added. The primary sequence of the predicted amino acid is shown in FIG.
It was shown to.

Purified hEGF.BF.H obtained in Example 3
After hydrolyzing CV-Env with 6N hydrochloric acid for 8 hours, PITC
The amino acid composition was measured by a method (PICO-TAG amino acid analysis system manufactured by Waters). The measured value of the amino acid composition is the expected hEGF-BF-HCV-E
The results are shown in Table 7 below together with the amino acid composition (theoretical value) determined from the primary sequence of the nv amino acid (FIG. 17). Both values were almost the same. Also, hEGF, BF, HCV-E
nv N-terminal amino acid sequence by Edman degradation (P. Ed.
man, Arch. Biochem. Biophy
s. , 22 , 475 (1949)) are shown in Table 8 below. The results were completely consistent with the expected N-terminal amino acid sequence (FIG. 17).

[0054]

[Table 7]

[0055]

[Table 8]

Further, hEGF / BF / HCV-Env
Of the C-terminal amino acid sequence of C.p.a. using carboxypeptidase Y (RP Ambler, Mehtod)
in Enzymology, 25 , 143 (197)
Table 8 shows the results examined in 2)). This also completely coincided with the expected C-terminal amino acid sequence of hEGF.BF.HCV-Env (FIG. 17). based on the above results,
The obtained hEGF.BF.HCV-Env was confirmed to be the target protein.

Next, by the same operation as described above, the amino acid composition, N-terminal and C-terminal of the purified HCV-Env obtained in Example 3
The terminal amino acid sequence was examined. The measured values of the amino acid composition are shown in Table 9 below together with the theoretical values obtained from the primary sequence of the predicted amino acid (FIG. 18). Both values were almost the same.

[0058]

[Table 9]

The N-terminal and C-terminal amino acid sequences are shown in Table 10 below. Both the N-terminal and C-terminal amino acid sequences were completely identical to the expected primary amino acid sequence (FIG. 18). From the above results, the obtained HCV-E
nv was confirmed to be the target protein.

[0060]

[Table 10]

[0061]

According to the present invention, a high expression vector derived from Bacillus brevis has been developed. Since this high expression vector can freely insert a foreign gene of interest and can express it at a high level, by culturing a microorganism transformed with this vector, various types of foreign gene products of interest can be obtained. Can be secreted and produced in and out of cells.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence encoding the amino acid sequence of hEGF.

FIG. 2 is a restriction map of the expression vector pHT · hEGF.

FIG. 3 is a restriction map of the expression vector pHT.hEGF.R.

FIG. 4 is a restriction map of the expression vector pHT.hEGF.RX.

FIG. 5 shows a base sequence ST.

FIG. 6 shows a base sequence BF.

FIG. 7: hEGF containing a part of the MWP signal peptide
1 shows a synthetic DNA encoding the amino acid sequence of

FIG. 8 is a construction diagram of plasmid pHT110 · hEGF.

FIG. 9 is a construction diagram of plasmid pHT · hEGF · ST.

FIG. 10 shows the nucleotide sequence of a synthetic IGF-I gene (including restriction enzyme sites added to the N-terminal and C-terminal).

FIG. 11: Plasmid pHT / hEGF / ST / IGF
It is a construction diagram of -I.

FIG. 12 shows the primary amino acid sequence of hEGF.ST.IGF-I.

FIG. 13 shows the primary sequence of the amino acids of IGF-I.

FIG. 14 is a construction diagram of a plasmid pHT.hEGF.BF.

FIG. 15 shows the nucleotide sequence of a synthetic HCV-Env gene (including restriction enzyme sites added to the N- and C-termini).

FIG. 16: Plasmid pHT / hEGF / BF / HCV
It is a construction diagram of -Env.

FIG. 17 shows the primary amino acid sequence of hEGF.BF.HCV-Env.

FIG. 18 shows the primary amino acid sequence of HCV-Env.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification symbol FI (C12P 21/02 C12R 1:08) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 15/75 C12N 1 / 21 C12P 21/02 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. The amino acid sequence of SEQ ID NO: 1 in the sequence listing
That has the base sequence to be loaded,
As shown in the restriction map of FIG.Bacillus Brevis
(Bacillus brevis HP926(F
ERM BP-5382)
Expression vector pHT · hEGF. 2. The restriction enzyme map shown in FIG.
3. An expression vector pHT.hEGF.R represented by the restriction map shown in FIG. 3 below, wherein a base sequence R encoding a protease-recognizing amino acid sequence is bound to the expression vector pHT.hEGF described in 1.). 3. The method according to claim 2, wherein the restriction map is shown in FIG.
4. An expression vector pHT.hEGF.R.X represented by the restriction map shown in FIG. 4 below, comprising a foreign gene X bound to the expression vector pHT.hEGF.R described in 1. 4. The expression vector pHT according to claim 3,
A microorganism having hEGF.R.X. 5. The expression vector pHT according to claim 3,
Bacillus brevis owning hEGF-RX (Ba
C. brevis). 6. The expression vector pHT.
Bacillus brevis H10 holding hEGF ・ RX
2 (Bacillus brevis H102). 7. The expression vector pHT according to claim 3,
A method for producing a gene product, comprising producing and accumulating a foreign gene product in a culture by culturing a microorganism having hEGF.R.X, and collecting the product.