JPS61166400A - Protein production - Google Patents

Abstract

PURPOSE:A bacterium in Bacillus is transformed with a recombinant DNA and the resultant transformed strain is cultured to enable easy production of a desired protein such as human beta-interferon which has required much labor force for conventional production. CONSTITUTION:An expression and excretion vector bearing the DNA base sequence including the zone participating to the expression of exoenzyme genes which are massively produced in a microorganism in Bacillus and to the excretion of the protein resultantly produced is linked with a gene for a desired protein to give a recombinant DNA. Then, the recombinant DNA is introduced into a microorganism in Bacillus as a host to allow the microorganism to produce the desired protein. The above-stated expression and excretion vector means a combination of the DNA base sequence including the zone participating to the expression of gene and the excretion of the protein which is resultantly produced, with a phase or plasmid replicable in a microorganism in Bacillus.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for secreting and producing a protein produced by a Bacillus bacterium using recombinant DNA into the culture solution of the bacterium.

The present invention particularly relates to an expression/secretion vector having a DNA base sequence containing a region involved in the expression of an exoenzyme gene that is secreted and produced in large quantities in bacteria of the genus Bacillus and the secretion of the resulting protein, and a desired protein. The present invention relates to a method for causing a Bacillus bacterium used as a host bacterium to secrete and produce a desired protein by introducing a recombinant DNA obtained by linking the genes of the present invention into a bacterium belonging to the genus Bacillus.

Regions involved in gene expression include the promoter region, which includes the -35 and -10 regions that are recognized and bound by RNA polymerase, and the DNA base sequence that allows mRNA synthesized by RNA polymerase to bind to ribosomes. There is a coding region for bosomal binding.

In addition, in order to secrete the protein produced as a result of expression from the bacterial body to the outside of the bacterial cell, a polypeptide called a secretion signal that is present upstream of the mature protein that exists outside the bacterial cell is essential. This polypeptide is synthesized within the bacterial body in a form linked to the amino terminal upstream of the mature protein, and it has been revealed that it plays an important role in passage through the cell membrane of the bacterial cell during secretion (G, Blobel J, Ce1l, Biol
67835 (1975)).

Therefore, the region encoding the polypeptide is an essential region for secreting the expressed protein.

With the remarkable development of molecular biology in recent years, it has become possible to secrete and produce a desired protein by ligating the gene encoding the desired protein with an appropriate vector and introducing the protein into the host bacterium.

Although any bacteria can be used as the host bacteria, for example, when Escherichia coli is used, the desired protein usually accumulates within the bacterial body, making it extremely difficult to purify the protein. Contamination with pyrogenic substances is unavoidable and has become a major problem. On the other hand, Bacillus bacteria have the property of secreting a large amount of protein to the outside of their cells, are nonpathogenic, and have been used in the fermentation industry for a long time (Debabov, The Molecule
ar Biology of the Bacilli“
1332 (19B2)) Dubnow, D.A., ed. Academic Press) is widely known, and from the viewpoint of protein production by microorganisms, creating expression and secretion vectors that can be used in Bacillus bacteria is a major industrial endeavor. It is of great significance. ~ Hereinafter, the present invention will be explained in detail.

The expression/secretion vector in the present invention refers to a vector in which a DNA base sequence containing a region involved in gene expression and secretion of the protein produced as a result is combined with a phage or plasmid that can be replicated in Bacillus bacteria. Expression/
By ligating a DNA base sequence encoding a desired protein to a secretion vector downstream of a region involved in desired expression and secretion of the resulting protein, the desired protein is introduced into a Bacillus genus bacterium that serves as a host bacterium. A gene encoding a gene is expressed in a host bacterium, and the resulting protein is secreted into the culture solution of the host bacterium in large quantities and can be recovered and purified from the culture supernatant in a simple process. It is.

Any plasmid or phage constituting the expression/secretion vector can be used as long as it can be replicated in Bacillus bacteria, but a commonly used plasmid is Staphylococcus nopUB110°. From the downstream part of the DNA base sequence involved in expression and secretion, the DNA is designed to retain the region involved in gene expression and secretion of the protein produced as a result.
Hydrolyze (digest) the phosphodiester bond of the A base sequence
A DNA base sequence having an appropriate restriction enzyme cleavage site is linked downstream of the region involved in expression and secretion of the protein produced as a result, thereby creating a DNA base sequence that defines the desired protein. Expression/secretion vectors of the present invention also include expression/secretion vectors that can be easily linked directly or using an appropriate linker. Any nuclease may be used to digest the DNA base sequence so as to preserve the DNA base sequence including the region involved in gene expression and secretion of the protein produced as a result, but exonuclease Ba131 is usually used. It is convenient to use

When a desired protein gene is expressed using the expression/secretion vector using a Bacillus bacterium as a host strain, the RNA polymerase and ribosome of the Bacillus bacterium have strict specificity regarding recognition of the promoter region and ribophum binding region. Because it has (Sueharu Horinouchi, “Proteins, Nucleic Acids, Enzymes,” 1468 (1983)) Those regions are
It must be from the genus Bacillus (Goldf
arb, D, S, etat Nature 2
93309 (1981)).

Furthermore, from a practical point of view, when creating an expression/secretion vector, it is desirable to derive it from an extracellular enzyme gene that can be secreted and produced in large quantities outside the bacterial cell in a short period of time in terms of large-scale expression and secretory production. .

Neutral protease, alkaline protease, α-amylase, Lebanese sucrase, etc. are known as extracellular enzymes of bacteria belonging to the genus Bacillus. Focusing on the fact that the neutral protease gene is secreted in large quantities, we determined the entire DNA sequence of the neutral protease gene, including the batium binding region and the region involved in the secretion of the expressed protein. Gansho 59-1751
58).

Based on these findings, exonucleases are used to cleave the gene from the restriction enzyme cleavage site downstream of the DNA base sequence that includes the region involved in the expression of the neutral protease gene and the secretion of the resulting neutral protease gene. The DNA is constructed in such a way that the DNA base sequence containing the regions involved in expression and secretion of the resulting protein is preserved.
After digesting the base sequence, for example, by preparing a recombinant DNA to which the human β-interferon gene has been linked, and introducing the recombinant DNA into Bacillus subtilis, human β-interferon is introduced into the medium. We succeeded in secreting production.

Furthermore, a DNA base sequence having a restriction enzyme cleavage site shown in Figure 4 was placed downstream of a DNA base sequence having a region involved in gene expression and secretion of the protein produced as a result, and was replicated in Bacillus bacteria. We succeeded in constructing an expression/secretion vector combined with a possible plasmid.

That is, the expression/secretion vector uses an exonuclease to express the gene from a restriction enzyme cleavage site located downstream of the DNA base sequence that has a region involved in the expression of the neutral protease gene and the secretion of the protein produced as a result. After digesting the DNA base sequence so that the region involved in the secretion of the protein produced as a result is retained,
This is a combination of the DNA base sequences shown in FIG. By using this expression/secretion vector, the restriction enzyme cleavage site in the DNA base sequence shown in Figure 4 can be used to join the gene of the desired protein, making it very convenient for practical use. be.

From this, using the expression/secretion vector of the present invention,
Transform Bacillus subtilis using recombinant DNA of a desired gene, such as interferon growth hormone, interleukin, nerve growth factor, kallikrein, or plasmino, at the restriction enzyme cleavage site present in the DNA base sequence shown in Figure 4. The expression of the human β-interferon gene and the secretory production of the human β-interferon produced as a result of the conversion will be described. Note that the present invention is not limited in any way by the following examples.

Plasmid pNP150 containing the enzyme gene was transferred to Bacillus subtilis MT-0150 (FERM-BP-42).
5) and cut it open with the restriction enzyme Pvu (according to a conventional method). Exonuclease Bal 31 was applied to the obtained linear plasmid, and the base pairs of about 220 BL from both ends of the above linear plasmid were extracted. was removed. The restriction enzyme Dp from pIF20 shown in Example 5 was added to the cleavage site.
A recombinant DNA was prepared in which the promoter obtained by cleaving with n2 and the gene encoding human β-interferon from which the secretory signal region had been removed were ligated. Using Bacillus subtilis IA310 (Ohio University Bacillus stock center) as a host,
The strain was then grown in BY solution supplemented with 0.2% NaCl, 1% polypeptone, and 5 μg/rnl of kanamycin. Cultures were grown at 30°C with shaking.

After about 14 hours, the cultured bacteria was i o, o o o! i is 5
The mixture was centrifuged for a minute and the supernatant was collected. The plasmid immunized with human β-interferon activity contained in the supernatant by the EIA method was named pOZ361.

As a result of transforming the IA310 strain again using this plasmid, it was confirmed that the obtained kanamycin-resistant strain was a total teinterferon-producing strain. Creation of Example 2 Plasmid DNA pTUB 4 (Takeic
hIetat Agric Biol, Chem 47
159-161 (1983)) 10μg of restriction enzyme A
1u ■ (Takara Shuzo) 10 units and restriction enzyme EcoRI
(manufactured by Takara Shuzo) and reacted at 37° C. for 1 hour. The composition of the reaction system was 10mM Tris-HCl buffer (p
H7,5), l OmM Hlci, .

150mM NacJ. The first half (approximately 400 base pairs) of the DNA fragment containing the α-amylase gene, which lacks the region involved in the expression of the gene obtained as a result of the reaction and the secretion of the protein produced as a result, was added to 1.0% low melting point agarose ( BRL Inc.) was separated by gel electrophoresis, and the DNA was extracted from the gel. The DNA was purified by phenol extraction and chloroform extraction, and then recovered by ethanol precipitation. The recovered DNA was diluted with 50mM
The DNA of the first half of the α-amylase gene, which lacks the region involved in gene expression and secretion of the protein produced as a result, was dissolved in 10μj of HCl buffer (pH 7,5).
fragment (hereinafter referred to as fragment A).

Plasmid pUc13 DNA (PL-Pharmacia Vina Chemical) was completely cleaved with restriction enzyme SmaI (Takara Shuzo) and restriction enzyme EcoR■ (Takara Shuzo).
After hydrolyzing the terminal phosphate ester using E. coli alkaline phosphatase (manufactured by Takara Shuzo), it was combined with fragment A using T4 ligase (manufactured by Takara Shuzo). pUC
The cleavage of No. 13 is 5 μy of pUC13 DNA.

-■ 10 units and EcoRl: 10 units were reacted at 37°C for 1 hour.

The composition of the reaction system is IQmM! JS hydrochloric acid buffer (pH 7,
5), 10mM Mgcl, 150mM Nacl.

Sma I and Ii obtained? pU cleaved with coR[
C13 DNA was sequentially purified by phenol extraction, ether extraction, and ethanol precipitation.

The obtained pUC13 DNA was 20ttl of 50mM-
The vector DNA was dissolved in 1-Lis-HCl buffer (pH 7.5). The vector DNA and fragment A were ligated using E. coli T41J gauze. The composition of the reaction system was fragment AIOμ, vector DNA10μ, and Escherichia coli T.
41J Gase 5 units 66rnM IJ-HCl buffer (pH 7,5) 6.6mM MgcJ, 10mM dithiothret-/u.

2mMATP (adenosine triphosphate).

The reaction was carried out at 15°C for 2 hours. -12 (JMIOI) to Escherichia coli using the recombinant DNA obtained as a result of the reaction.
strain (manufactured by BRL Inc.) as a host using a conventional method (Mande
l, M, and A, Higa, J, Mol, Bi.
Transformation was carried out according to OL 53154 (1970)). After transformation, add ampicillin to a final concentration of 50μi/m
TBAB medium (manufactured by Difco) added to
ampicillin-resistant strains were selected using

The obtained ampicillin-resistant strain was cultured in Penn Ansei medium (Di
After culturing at 37°C for 14 hours using fco fi) 50d,
Collect the bacteria, and remove the cells from the alkaline method (Birnboim).

H, C, et al Nucleic Ac1ds R
es, 71513 (1979)). The nucleotide sequence of the ligation site between plasmid pUC13 and the fragment is shown in FIG.

The obtained plasmid was digested with restriction enzyme BamH■ and restriction enzyme EcoRI, resulting in approximately 400 base pairs and approximately 27 base pairs.
A DNA fragment of 00 base pairs was found. This indicates that this plasmid has the SmaI cleavage site of pUc13 and the EC
This proves that this is a recombinant plasmid in which fragment A consisting of about 400 base pairs was inserted between the 0RI cleavage sites. Thereafter, this recombinant plasmid was used as pAM
It is abbreviated as ll.

Plasmid pAM11 DNA 50 tel contains the entire DNA base sequence of the first half of the α-amylase gene, lacking the restriction enzyme BamHI region. Next, 50 μg of the plasmid DNA (described above) containing the α-amylase gene derived from Bacillus subtilis was added with the restriction enzyme EcoR.
A DNA fragment (hereinafter referred to as fragment C) containing the latter half of the α-amylase gene consisting of about 1300 base pairs was prepared by cutting the DNA fragment with the restriction enzyme Pvu[I and the restriction enzyme Pvu[]. Furthermore, after digestion using restriction enzymes BamHI and H1ncII (manufactured by Takara Shuzo), plasmid pU012 DNA treated with E. coli alkaline phosphatase, fragment A, fragment B, and fragment C were ligated. The reaction was carried out using plasmid pUc12DN.
Using 5 μg of A, 110 units of HincI and BamHI 1
The temperature was 0 units at 37°C for 1 hour. Next, after phenol extraction and ethanol precipitation, E. coli alkaline phosphatase (5 units) and Tris-HCl buffer (pH 8,0) were added to give a final concentration of 0.1M.
The reaction was allowed to proceed for 02 hours. After the reaction was completed, the reaction solution was subjected to phenol extraction, ether extraction, and ethanol precipitation to recover DNA. Thereafter, it was redissolved in 20μ of 50mM Tris-HCl buffer.

1 μg of the thus obtained pUC12 DNA was ligated with Fragment B2μy and Fragment C2μ dissolved in Tris-HCl buffer (1) H7,5) using E. coli T 41J gauze (manufactured by Takara Shuzo) in the same manner as described above. -12 (JMIOI) to Escherichia coli using the obtained recombinant DNA.
The strain was transformed and a transformed strain showing resistance to ampicillin was obtained. Plasmid DNA was prepared from the obtained ampicillin-resistant strain by the alkaline method (described above). When the obtained plasmid DNA was treated with only the restriction enzyme EcoR■ and with the restriction enzyme BamHI and the restriction enzyme HindlI, it was found that the EcoR treatment resulted in cleavage at one site, resulting in approximately 4500
The base-paired DNA fragments are also combined with BamHI and Hind[[
(manufactured by Takara Shuzo), DNA fragments of about 1800 base pairs and about 2700 base pairs were confirmed, respectively. Combining the above results, the pU used as a vector
Since C12 is approximately 2700 base pairs, the obtained plasmid (hereinafter abbreviated as pAM29) is pUC1.
It was confirmed that this is a recombinant plasmid in which an α-amylase gene lacking the region involved in gene expression and secretion of the resulting protein was inserted between the B amHI cleavage site of 2 and the Hindl cleavage site. It was done. The connection site between the region containing the α-amylase gene of this plasmid pAM29 and pUC12 is shown in FIG. The part ζ upstream from the amine end of the α-amylase gene portion of pAM29 plasmid DNA is the restriction enzyme B a
There are cleavage sites for mHI, SmaI, and Sst■. Further, downstream from the carboxyl terminus, Hin
There are cleavage sites for dl[ and Pvu[, respectively.

Example 3 Gene expression using sonuclease and its Bacillus. amyloliquefaciens F strain (ATCC23350
) Plasmid containing the neutral protease gene derived from I)
NP150 DNA was obtained from Bacillus subtilis MT-0150 (FERM-BP-425) carrying the plasmid.
). The plasmid pNP150DNA10
μ, 9 was reacted with 10 units of restriction enzyme Pvu I (manufactured by Boehringer Mannheim) at 37° C. for 1 hour. The resulting pNP150 DNA cleaved with Pvu) was subjected to phenol extraction three times, residual phenol was removed by ether extraction, and then ethanol precipitation was performed and recovered.

The recovered DNA is then treated with exonuclease Ba131.
(Made by Boehringer Mannheim) 30°G50 in 10 units
The reaction was carried out for minutes. The composition of the reaction system is 20mM! - Lys-HCl buffer (pH 8,1), 1mM EDTA, 12m
MCacl! 2,12mM Mgcl! ,,600mMN
ac/. After the reaction was completed, phenol extraction, ether extraction, and ethanol precipitation were sequentially performed, and then the DNA was recovered. The recovered DNA (hereinafter abbreviated as nuclease-treated DNA) was dissolved in 50 μl of 50 mM Tris-HCl buffer (pH 7.5).

It was confirmed by 1% magarose gel electrophoresis that the nuclease-treated DNA was a mixture of DNA fragments cut into various lengths by exonuclease.

Example 4 295 μg of pAM shown in Example 2 was treated with restriction enzyme 5st
I (manufactured by BRL Inc.) 10 units, restriction enzyme Pvu [
(manufactured by Boehringer Mannheim) using 10 units, 37
The reaction was allowed to take place at °C for 1 hour. The resulting α-amylase gene (which lacks the region responsible for the expression of the approximately 2000 base pair gene and the secretion of the resulting protein)
Hereinafter, it will be abbreviated as α-amylase structural gene. ) was isolated, purified, and recovered using the same method as described above. Subsequently, both ends of the DNA were treated with E. coli T4 polymerase as described in Example 2. Since the DNA is a mixture of DNA fragments of various lengths, the recombinant DNA is a mixture of DNA fragments of various lengths. It forms a mixture. Using the obtained recombinant DNA mixture, Chang's protoplast method (Chang, S.
and Cohen, S.N., Mo1. Gen, Ge
Bacillus subtilis was transformed according to nert168 11.1 (1978)).

To the protoplast regeneration medium, 100 μI/ml of kanamycin sulfate (Boehringer, Mannheim fi) and soluble starch (manufactured by Wako Tsumugi Pharmaceutical) were added to a final concentration of 1%. The host bacteria used for transformation were Bacillus subtilis IA and Bacillus 289 (Ohio University, Bacillus.

The stock center stock) was used. Iodine was used to select transformed strains secreting and producing amylase.

Iodopotash method (J, Bacteriol 11941
6-424 (]974)) by starch decomposition to form 710-. As a result, several dozen transformed strains secreting and producing amylase were obtained. These transformed strains were cultured with shaking at 37° C. for 8 hours using BY medium, and the α-amylase activity present in the culture supernatant was measured. The activity was measured by a method of examining the production of reducing groups from soluble starch using dinitrosalicylic acid (BiochemicaInformat
ion II Boehringermann/%im p28-30)
Next, from the transformed strain that produces amylase,
Plasmids were prepared according to the alkaline method described above. Each of the obtained plasmids was treated with restriction enzyme B am
HI *Restriction enzyme Sma I + and restriction enzyme Sst
The plasmid was cut with BamHI and examined using 1% agarose gel electrophoresis.
It was found that I was cleaved at two places and 5stII was cleaved at two places.

The DNA portion of pNP 150 involved in the expression of the neutral protease gene and the secretion of the resulting protein was cleaved at only one site with the restriction enzyme 5stI [BamHI].

It is known that it is not cleaved by SmaI. Also α
- It is known that the amylase structural gene is cleaved at only one site with the restriction enzyme SmaI and not with the restriction enzymes BamHI and 5stII. Furthermore, α consisting of about 2000 base pairs obtained by treating with restriction enzyme 5stI and restriction enzyme Hind) prepared from pAN29.
- The amylase structural gene has been found to have a restriction enzyme Sma I site and a restriction enzyme Bam HI site immediately before its amino terminus.

From these results, the plasmid obtained from the transformed strain that secretes α-amylase contains the region necessary for the expression of the neutral protease gene and the secretion of the resulting protein and the DNA fragment obtained from pAM29. It was confirmed that the contained recombinant DNA was contained. This recombinant D
The connection site between the DNA portion involved in the expression of the neutral protease gene contained in NA and the secretion of the resulting protein and the α-amyl-7 structural gene is shown in FIG. A DNA encoding the desired protein is inserted into the restriction enzyme cleavage site of the DNA base sequence shown in FIG. 4 of the recombinant DNA.
This means that by inserting the A fragment, the ability to produce α-amylase is deleted, and a transformed strain that produces the protein can be easily selected. One of these recombinant DNAs was named pNPA74.

Example 5 A human β-interferon gene was prepared using plasmid pIF20 containing the gene.

The plasmid pIF20 contains the restriction enzyme 5au3 at both ends of the DNA base sequence encoding the mature protein from which the signal portion involved in the secretion of human β-interferon has been deleted.
The plasmid pIF20 was constructed using a synthetic N A IJ linker so that it had a restriction enzyme Dpn I cleavage site and a restriction enzyme Dpn I cleavage site.
When digested with u3A (manufactured by Takara Shuzo) or restriction enzyme Dpni (manufactured by Takara Shuzo), human-β-
A DNA base sequence encoding a mature protein (hereinafter abbreviated as human β-interferon structural gene) from which the signal portion involved in interferon secretion has been deleted can be prepared.

The human β-interferon structural gene (5 μl) was completely digested with the restriction enzyme BamHI into the expression/secretion vector pNPA74 (5 μl) and inserted using Escherichia coli T4 ligase to produce recombinant DNA. was used to transform amylase-deficient mutant Bacillus subtilis strain 1A289.As described in Example 3, the DNA fragment encoding the human-β-interferon structural gene was inserted into the BamHI cleavage site of pNPA74, as shown in FIG. The Bacillus subtilis IA289 strain transformed with the inserted recombinant DNA did not have amylase-producing ability and could be easily distinguished from the strain transformed with pNPA74.

The activity of human β-interferon contained therein was measured. The measurement method is an enzyme immunoassay using anti-human β-interferon serum (enzyme immunoassay, edited by Eiji Ishikawa, p.
67 (1981)) was used.

As a result, a plasmid was prepared for transformation using the alkaline method described above for human β-interferrolysis. When the obtained plasmid DNA was cut with the restriction enzyme PvuI, it was cut at two positions.

Human β-interferon structural gene is restriction enzyme Pv
It is known that uII causes cleavage at one location. Also,
It is known that pNPA74 DNA used as an expression/secretion vector is similarly cleaved at one site with the restriction enzyme PVuII. Based on these facts, the plasmid obtained from the strain producing human β-interferon is a recombinant DNA in which the human β-interferon structural gene is integrated into the expression/secretion vector pNPA74 of the present invention.
It was confirmed that A.

By using recombinant DNA, it has become possible to secrete and produce human β-interferon by Bacillus and subtilis very easily.

In addition, the lu (unit) unit shown here is lX10mg
This corresponds to human β-interferon.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the method for creating Bacillus subtilis O2361, Figure 2 is a restriction enzyme map of pOZ361, and Figure 3 is a diagram showing human β-interferon DNA and pNP150.
FIG. 4 shows the nucleotide sequence of the binding site of the restriction enzyme SmaI and the restriction enzyme Bam
FIG. 5 is a diagram showing a DNA base sequence having a HI cleavage site; FIG. 5 is a diagram showing a DNA base sequence of a joining site between plasmid pUc13 and fragment A; The figure shows the DNA base sequence of the region containing the α-amylase gene of plasmid pAM29 and the connection site with I) UC12, and the frame shows the DNA base sequence of the region containing the α-amylase gene. . In addition, in FIG. 4.5.6, A represents atenine, C represents cytosine, G represents guanine, and T represents thymine. Figure 7 is a diagram showing the method for creating plasmid pNA74, and Figure 8 is a diagram showing the method for creating plasmid pNPI74. In these figures, the white frame represents the neutral protease gene, and the hatched frame represents α. - amylase structural gene, 1 indicates a region involved in the expression of the neutral protease gene, and 2 indicates a region involved in the secretion of the neutral protease. Patent Applicant: Director of the Agency of Industrial Science and Technology, Tatsumi Todoroki 1. The moon changes to Yukushu (η name / @ I 1 and sends F 1 No. Kowani Ko 1 Kanpo 1: Yo 31 Sukusoni Shiku゛Turf L mouth〉) 8 + II 4 to Suf 9 -2〉7↓, proboscis'' day [-hard λL DIJA Hideyo F:・1i2shi-
M If! L no;, dl-1 rod 2 Figure vu11 GTGGGTTTAG GTMGAAATT GT
CTAGTGCTAGTCTGCCGG GTGTT
CAGGCCGCTGAGAATGTACCGMGCA
TTCTTTGGT GCMTCAGMAGCTAC
AACT TGCTTGGATT CCTACAA
AGA rod 3 mouths GTAGCCGCTT CCTTTATGAG T
TTAACCATCAGCAGCMTT TTCAG
TGTCA GMGCTCCTGF4 Figure 6

Claims (1)

[Scope of Claims] 1) A method for producing a protein, which is characterized in that a Bacillus bacterium is transformed using a recombinant DNA, the resulting transformed strain is cultured, and a desired protein is recovered from the culture supernatant. Production method. 2) The recombinant DNA consists of an expression/secretion vector that has a DNA base sequence that includes a region involved in gene expression and secretion of the protein produced as a result, and a DNA base sequence that encodes the desired protein. A method for producing a protein according to claim 1, characterized in that: 3) An expression/secretion vector in which the recombinant DNA has a DNA base sequence containing a region involved in the expression of the extracellular enzyme gene of a Bacillus bacterium and the secretion of the extracellular enzyme protein produced as a result, and the desired protein. DNA that codes for
Claim 1 consisting of a base sequence
The method for producing the protein described in section. 4) An expression/secretion vector in which the recombinant DNA has a DNA base sequence containing a region involved in the expression of the extracellular neutral protease gene of a Bacillus bacterium and the secretion of the neutral protease produced as a result, and the desired protein. The method for producing a protein according to claim 1, characterized in that the method comprises a DNA base sequence that defines the protein. 5) DNA whose DNA base sequence, which includes a region involved in gene expression and secretion of the resulting protein, is derived from a plasmid or phage that can be replicated in Bacillus bacteria.
The expression/secretion vector according to claim 2, which is linked to the A base sequence. 6) An expression/secretion vector characterized in that the extracellular neutral protease gene according to claim 4 is derived from Bacillus amyloliquefaciens. 7) D containing a restriction enzyme cleavage site capable of binding a DNA base sequence containing the gene of the desired protein and a region involved in gene expression and secretion of the protein produced as a result.
3. The expression/secretion vector according to claim 2, which has an NA base sequence downstream. 8) From the restriction enzyme cleavage site located downstream of the DNA base sequence containing the region involved in the expression of the extracellular enzyme gene of Bacillus bacteria and the secretion of the extracellular enzyme protein produced as a result, the expression of the gene and It is constructed by digesting the DNA sequence containing the gene so that the DNA sequence containing the region involved in the secretion of the resulting protein is retained, and then inserting and ligating the DNA sequence of the desired protein. The expression/secretion vector according to claim 2, which is characterized in that: 9) Expression/secretion vector according to claim 8, characterized in that an exonuclease is used when digesting the DNA base sequence. 10) Expression of extracellular neutral protease gene of Bacillus genus and its results. From the restriction enzyme cleavage site located downstream of the DNA base sequence containing the region involved in the secretion of the extracellular neutral protease produced, exonuclease is used to express the gene and secrete the protein produced as a result. After digesting the DNA base sequence containing the gene so that the DNA base sequence containing the region containing the gene is retained, the DNA having a restriction enzyme cleavage site that can bind to the desired protein gene is digested.
The expression/secretion vector according to claim 2, which is constructed so that the base sequence is present. 11) The expression/secretion vector according to claim 11, wherein the DNA base sequence having a restriction enzyme cleavage site is included in the following. 5'CGCCCGGGGATCCCCC 3'3'GCG
GGCCCCTAGGGG 5'