JP2500311B2 - Protein production method - Google Patents

Description

The present invention relates to a method of secreting and producing a desired protein into a culture supernatant of a bacterium of the genus Bacillus as a host bacterium by a genetic engineering technique.

The present invention particularly relates to a DNA nucleotide sequence involved in the expression of extracellular neutral protease which is secreted and produced in large amounts in Bacillus bacteria, and a DNA encoding the pre-pro structure of the protease.
Introduction of a recombinant DNA obtained by ligating an expression / secretion vector having a nucleotide sequence with a DNA nucleotide sequence containing a gene of a desired protein so as to allow expression and secretion into a Bacillus bacterium used as a host bacterium The present invention relates to a method for secreting and producing a desired protein in the bacterium.

DNA involved in the expression of extracellular neutral protease gene
As a nucleotide sequence, it is important that a promoter region containing −35 and −10 regions, which are regions recognized and bound by RNA polymerase, and a ribosome binding region for binding mRNA synthesized by RNA polymerase to ribosome. Are known. And not only the DNA base sequence of these regions, the number of nucleotides between these regions,
The number of nucleotides from the ribosome binding region to the translation start site is also known to be important. In addition, the extracellular neutral protease produced as a result of expression has a polypeptide called a signal structure as a region necessary for being secreted out of the extracellular body.

The polypeptide is also called a pre-structure and is bound to the N-terminal side of the polypeptide existing upstream of the N-terminal of the mature extracellular neutral protease and deleted during secretion, that is, to the N-terminal side of the pro-neutral protease. It is known that it plays an important role in cell membrane passage during secretion (G. Blobel JC
ell Biol 67 835 (1975)).

The signal structure and pro structure as described above exist as a single polypeptide called a pre-pro structure, and the structure is not present in the secreted mature extracellular neutral protease. Although it is considered that they are sequentially cleaved, it is easy to understand that this cleavage process is associated with efficient secretion of neutral protease.
Therefore, it can be said that the DNA base sequence encoding the pre-pro structure is an important region for secreting the expressed extracellular neutral protease.

The DNA base sequence encoding the pre-pro structure of the present invention can represent, for example, the 252nd to 914th bases counted from the 5 ′ end upstream of the DNA base sequence shown in FIG.

DNA encoding the pre-pro structure of the extracellular neutral protease gene of Bacillus amyloliquefaciens
The nucleotide sequence contains the 276th to 278th "AG
The sequence corresponding to "T" (encoding serine) is "GTT".
There are allelic variants substituted for (encoding valine), and such a DNA base sequence encoding the pre-pro structure of the present invention is also included.

From the above, an expression / secretion vector having a DNA base sequence involved in the expression of an extracellular neutral protease gene and a DNA base sequence encoding a pre-pro structure was produced as a result of expressing a heterologous gene. It has an important meaning in secreting the protein of the above.

Recent remarkable progress in genetic engineering has made it possible to produce heterologous proteins using microorganisms. However, since Escherichia coli, which is usually used as a host bacterium, accumulates a desired protein in the microbial cells, the production amount of the protein is limited accordingly, and it is possible to separate the desired protein from other microbial proteins during purification. It was very difficult.

Further, the purified desired protein is often contaminated with a pyrogen derived from Escherichia coli, and in these respects, genetically engineered substance production using Escherichia coli as a host poses a serious problem from the viewpoint of practical use. It is a thing.

On the other hand, Bacillus bacteria have no pathogenicity and have been used for a long time in the production of antibiotics, extracellular enzymes or nucleic acid-based seasonings by exocrine secretion. Creating a secretion vector has great industrial significance from the viewpoint of substance production by microorganisms.

 The present invention will be described in detail below.

The Bacillus bacterium used as a host bacterium of the recombinant DNA of the present invention includes Bacillus subtilis, Bacillus,
Any one such as Amyloliquefaciens, Bacillus cereus, Bacillus megaterium, Bacillus stearothermophilus, etc. can be easily used for Bacillus subtilis which has been genetically well studied.

Any method may be used for transformation of the host bacterium, but when Bacillus subtilis is used as the host, the protoplast method (S.
Chang, SNCohen, Mol.Gen.Genet.1979, 168, 111) and a competent method (C.Anagnostopouls, J.Spizizen, J.Bact
eriol.1961, 81 , 741) is an effective method.

The expression / secretion vector in the present invention is a DNA involved in the expression of extracellular neutral protease gene of Bacillus.
Refers to a combination of a nucleotide sequence and a DNA nucleotide sequence encoding the pre-pro structure of the neutral protease and a phage or plasmid capable of replicating in the genus Bacillus or a derivative thereof, and is a desired protein for the expression / secretion vector. The desired protein can be secreted and produced outside the cell by ligating a DNA base sequence containing the gene encoding the protein.

That is, a DNA base sequence containing a gene for a desired protein is ligated downstream of a DNA base sequence encoding a pre-pro structure of a neutral protease contained in the expression / secretion vector, and introduced into a Bacillus bacterium used as a host bacterium, It is possible to transform the bacterium to express a gene encoding a desired protein in a host bacterium and secrete the desired protein produced as a result of the bacterium.

The desired protein secreted in this manner is recovered by removing the cells from the culture solution by centrifugation or centrifugation, and applying a usual method for purifying extracellular enzymes and the like from the obtained supernatant.

This is a particularly advantageous point from the viewpoint of industrial substance production, since the purification process can be greatly simplified as compared with the case where a desired protein is accumulated in the cell body such as Escherichia coli.

Any plasmid, phage, or derivative thereof constituting the part involved in replication of the expression / secretion vector can be used as long as it can be replicated in the bacterium of the genus Bacillus used as a host. Those having a gene involved in substance resistance are convenient for selecting transformants. As a plasmid that is often used, a plasmid derived from Staphylococcus
Examples of pUB110, pTP5, pUC194, pE194, pSA0501, pBD6 and the like, and examples of phages include φ15, φ29, φ105 and ρ11.

As described above, the expression / secretion vector of the present invention has a DNA base sequence involved in the expression of extracellular neutral protease, and expresses a gene of a desired protein by this DNA base sequence. -The same polypeptide as the pre-pro structure of the neutral protease is added to the N-terminus of the desired protein depending on the DNA sequence encoding the pro structure.

The presence of the polypeptide enables the secretion of the desired protein to the outside of the Bacillus bacterium used as a host bacterium.

Therefore, in the expression / secretion vector, a DNA base sequence including a region encoding a desired protein must be linked immediately downstream of a DNA base sequence involved in expression and a DNA base sequence encoding a pre-pro structure.

Such a bond is a restriction existing near the cleavage point of the DNA base sequence encoding the N-terminal portion of the mature enzyme that is finally cleaved when the extracellular neutral protease is secreted, that is, the cleavage point portion of the pro structure. This is accomplished by using an endonuclease cleavage site.

Especially in the case of the extracellular neutral protease gene of Bacillus amyloliquefaciens, there is a cleavage site for the restriction endonuclease SphI in the DNA base sequence that encodes the vicinity of the pro-structure cleavage point. A DNA base sequence containing the coding region can be bound.

That is, a recombinant DNA necessary for secreting a desired protein can be constructed by directly or indirectly using a linker or the like ligating a DNA base sequence containing a region encoding the desired protein to the SphI cleavage site. . Therefore, instead of directly binding the DNA base sequence containing the region encoding the desired protein to the SphI cleavage site, a DNA base sequence having one or several restriction endonuclease cleavage sites is bound to DN containing the protein coding region
Naturally, an expression / secretion vector of the type in which the A nucleotide sequence is bound is also within the scope of the present invention.

DN with such a restriction endonuclease cleavage site
The present inventors constructed a nucleotide sequence containing the nucleotide sequence shown in FIG. 1 as the A nucleotide sequence. This nucleotide sequence was obtained by cleaving the SphI cleavage site near the pro structure cleavage point with the enzyme and then using an enzyme having an exonuclease activity. After removing the protruding ends to make them blunt ends, DNA base sequences including the base sequence shown in FIG. 1 are bound.

This DNA sequence has restriction endonucleases SmaI and B
Since the cleavage site of amHI exists, it is convenient for binding a DNA base sequence containing a region encoding a desired protein (Fig. 2).

Further, the present inventors have shown that the DNA base sequence containing the α-amylase gene of Bacillus bacterium (which has deleted the DNA base sequence encoding the original secretion signal) is bound to the downstream of the DNA base sequence shown in FIG. Recombinant DNA was obtained by ligating the DNA base sequence of Escherichia coli to the blunt end of the SphI cleavage site near the prostructure cleavage point of the extracellular neutral protease gene as described above (FIG. 5).

It encodes a DNA nucleotide sequence and a pre-pro structure involved in the expression of extracellular neutral protease gene.
Immediately downstream of the NA base sequence pro structure, a DNA base sequence containing the α-amylase gene bound to the DNA base sequence shown in FIG. 1 was bound.

By introducing this recombinant DNA into a bacterium belonging to the genus Bacillus, the α-amylase gene is expressed by the DNA nucleotide sequence involved in the expression of extracellular neutral protease, and the pre-pro structure of the extracellular neutral protease is expressed as N. -Pre-pro α-amylase added upstream of the terminal is synthesized in the cells.

In this case, the expression substance is secreted out of the cells by the action of the pre-pro structure, but at that time, the pre-pro structure is cleaved and accumulated in the medium in the form of mature α-amylase. That is, when it is considered that α-amylase is a desired protein, such recombinant DNA becomes a recombinant DNA that can be used when producing the protein. The DN shown in Fig. 1 is placed between the α-amylase gene and the SphI cleavage site near the cleavage point of the pro structure with blunt ends.
A base sequence exists. Therefore, a DNA base sequence containing a region encoding a new desired protein can be bound by utilizing the cleavage sites of SmaI and BamHI existing in the DNA base sequence.

That is, this recombinant DNA can also be called an expression / secretion vector. In particular, this expression / secretion vector allows the host bacterium to produce α-amylase when the vector alone is used, but once the DNA base sequence containing the region encoding the desired protein was inserted and ligated into the aforementioned SmaI or BamHI cleavage site. In some cases, the production and secretion of α-amylase present in the vector is stopped.

This means that the process can be extremely simplified in selecting a transformant having a recombinant DNA in which a DNA nucleotide sequence containing a region encoding a desired protein of interest is linked to an expression / secretion vector. .

That is, a bacterium that forms a halo by an iodine-starch reaction on a starch-containing agar medium contains the expression / secretion vector itself, but a bacterium that does not form a halo encodes the desired protein in the expression / secretion vector. It shows that the DNA base sequence containing the region is inserted and bound.

The present inventors succeeded in secreting and accumulating various desired proteins extracellularly using the expression / secretion vector of the present invention. This means that using the expression / secretion vector, various proteins such as interferon, growth hormone, interleukin, nerve growth factor, kallikrein, plasminogen activator or industrial enzyme are secreted and produced extracellularly using Bacillus subtilis as a host. It shows that it has become possible to do so and has great significance from the viewpoint of industrial microbial production.

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

Example 1. Construction of expression / secretion vector A plasmid pNP150 containing a neutral protease gene of Bacillus amyloliquefaciens was prepared from Bacillus subtilis MT-0150 (FERM BP-425) by a conventional method. Digestion of the plasmid with the restriction endonuclease SphI gives a large fragment of about 5.4 kb and a small fragment of about 1 kb.

This large fragment was separated and recovered by low melting point agarose gel electrophoresis. The recovered DNA was purified by column chromatography, phenol extraction, ether extraction, and ethanol precipitation according to a conventional method.

0.1 μg of the purified DNA was ligated with 5 units of T ₄ ligase to give a circular plasmid. Coupling reaction composition is 66m M Tris-HCl buffer _{(pH7.5), 6.6m M MgCl 2} , 10m M dithiothreitol, a 2m M ATP. The reaction was carried out at 15 ° C for 3 hours.

Bacillus subtilis 1A510 using this reaction mixture
(Ohio University Bacillus Stock Center stock strain) was transformed by the protoplast method.

The resulting kanamycin-resistant transformant (MT-1150 (FER
Plasmids were extracted from M BP-722) by the alkaline method and physical maps were prepared using various restriction endonucleases.

As a result, the obtained plasmid pES150 was a DNA nucleotide sequence involved in the expression of the extracellular neutral protease gene of Bacillus amyloliquefaciens and a DNA nucleotide sequence encoding a pre-pro structure (claim 3 DNA base sequence described in the paragraph) and D near the breakpoint of the pro structure.
It was confirmed that the plasmid had a form in which the DNA nucleotide sequence encoding the mature extracellular neutral protease downstream of the SphI cleavage site present in the NA nucleotide sequence was deleted.

As shown in FIG. 2, this plasmid has a unique SphI cleavage site, and the DNA base sequence containing the region encoding the desired protein is directly or indirectly linked to the site by using a linker or the like to detect the codon. It is an expression / secretion vector that can be easily constructed to form a recombinant DNA used for secretory production of a desired protein if ligated with no displacement.

Furthermore, 1 μg of pES150 was cleaved with SSphI to open the ring, and then the protruding end was removed using the exonuclease activity of T ₄ polymerase, and the DNA base sequence shown in FIG. Expression shown in the figure
Secretion vectors pSB150 and pBS150 were constructed.

This expression / secretion vector has a D sequence shown in Fig. 1 downstream of the DNA sequence encoding the pro-structural breakpoint of neutral protease.
The NA base sequences are linked, and there are cleavage sites for the restriction endonucleases SmaI and BamHI in the sequences.

Therefore, if a DNA base sequence containing a region encoding the desired protein is linked to either of the cleavage sites directly or indirectly by using a linker or the like so as not to shift the reading of the codon, the desired protein is secreted and produced. Recombinant DNA used for this can be easily constructed.

The construction of pSB150 and pBS150 from pES150 is described below.

1 μg of pES150 was cleaved and opened with SphI according to a conventional method. The linear pES150 obtained was purified by phenol extraction, ether extraction and ethanol precipitation. T ₄ polymerase buffer 4μl of 10-fold concentration was dissolved in distilled water of the ones after drying 32 [mu] l, and incubated at 37 ° C. by adding 2m M αNTP 2μl and T ₄ DNA polymerase (Takara Shuzo) 3 units.

150μl of DNA buffer after 30 minutes (10 m M Tris - HCl buffer pH8.0,10m M Kcl, 0.1m M EDTA) were added phenol extraction, ether extraction, the DNA was purified perform ethanol precipitation. The DNA is tris dried 50 [mu] l - HCl buffer (50 m M, pH 7.5) was subjected to 10μl out and dissolved in the following operations.

That is, to 10 μl of this DNA, 0.2 μg of the DNA base sequence shown in FIG. 1, which was separately prepared by a synthetic method, was added and a ligation reaction was performed with T ₄ ligase.

Reaction system T ₄ ligase (Takara Shuzo) 20 units, 66m M Tris-HCl - buffer _{(pH7.5), 6.6m M MgCl 2} , 10m M dithiothreitol, at 2m M ATP, reaction 20 hours at 4 ° C. I did.

The total volume of the reaction solution was 50 μl.

Using 20 μl of the reaction solution thus obtained, Bacillus subtilis 1A510 was transformed by the protoplast method. A plasmid was prepared from the obtained kanamycin-resistant strain and a physical map was prepared according to a conventional method. The resulting plasmid had no SphI cleavage site and had one SmaI and one BamHI cleavage site. It was

There are two types of this, depending on the insertion direction of the DNA base sequence shown in FIG. 1, and they were named psB150 and pBS150, respectively.

Example 2 Secretion production of α-amylase by expression / secretion vector pES150 1) Preparation of α-amylase gene in which a DNA base sequence involved in expression and secretion was deleted Plasmid DNA pTUB4 (Takeichi) containing Bacillus subtilis-derived α-amylase gene etal Agric.Biol.Ch
em. 47 159-161 (1983)) 10 μg of restriction endonuclease AluI (manufactured by Takara Shuzo) and restriction endonuclease
A reaction was performed with 10 units of EcoRI (Takara Shuzo) at 37 ° C. for 1 hour.

The composition of the reaction system is 10 mM Tris-HCl buffer (pH 7.5), 1
0m M MgCl _2, is a 150m M Nacl. The first half (about 400 base pairs) of the DNA fragment containing the α-amylase gene lacking the region involved in the expression of the gene obtained as a result of the reaction and the secretion of the resulting protein was digested with 1.0% low melting point agarose (BR
L, Inc.) was separated by gel electrophoresis and the DNA was extracted from the gel.

The DNA was purified by phenol extraction and chloroform extraction, followed by ethanol precipitation to recover.

10 μl of the recovered DNA in 50 mM Tris-HCl buffer (pH 7.5)
The DNA fragment was dissolved in the above and was used as the DNA fragment of the first half of the α-amylase gene lacking the region involved in the expression of the gene and the secretion of the resulting protein (hereinafter referred to as fragment A).

Plasmid pUC13DNA (PL-Pharmacia Biochemical) is completely cleaved with restriction endonuclease SmaI (Takara Shuzo) and restriction endonuclease EcoRI (Takara Shuzo), and E. coli alkaline phosphatase (Takara Shuzo)
After the terminal phosphoric acid ester was hydrolyzed using, the fragment A was ligated with T ₄ ligase (Takara Shuzo).

pUC13 is cleaved by pUC13 DNA 5 μg, SmaI 10 units, EcoR
It was carried out by carrying out the reaction at 37 ° C. for 1 hour with 10 units of I.
The composition of the reaction system, 10 m M Tris-HCl buffer (pH 7.5), 10 m
A M MgCl _2, 150m M NaCl.

The resulting pUC13 DNA cleaved with SmaI and EcoRI was sequentially purified by the operations of phenol extraction, ether extraction and ethanol precipitation.

The resulting pUC13DNA is, 20 [mu] l of 50 m M - it was dissolved in Tris-HCl buffer (pH 7.5), and with a vector DNA.

Ligation of the vector DNA and fragment A was performed using E. coli T ₄ ligase. The composition of the reaction system was as follows: 10 μl of fragment A (buffer), 10 μl of vector DNA (buffer), E. coli T ₄ D
NA ligase 5 units 66m M Tris - HCl buffer (pH 7.5),
6.6m M MgCl ₂ 10m M dithiothreitol, a 2m M ATP (adenosine triphosphate). The reaction was carried out at 15 ° C for 2 hours.

Using the recombinant DNA obtained as a result of the reaction, E. coli K-1
2 (JM101) strain (manufactured by BRL Inc) as a host (Mandel.
Transformation was performed according to M. and A. Higa, J. Mol. Biol 53 154 (1970)). After transformation, ampicillin was added to a final concentration of 50.
Ampicillin-resistant strains were selected using TBAB medium (manufactured by Difco) added to give μg / ml.

The obtained ampicillin-resistant strain was transferred to a pen assay medium (Di
After culturing at 37 ° C. for 14 hours using 50 ml of Fco), the cells were collected, and the cells were collected by the alkaline method (Birnboim.HC et al Nucleic Acids R
The plasmid was prepared according to es 7 1513 (1979).

The nucleotide sequence at the junction between plasmid pUC13 and fragment A is
This is shown in FIG. The resulting plasmid was digested with the restriction endonucleases BamHI and EcoRI,
DNA fragments of 400 base pairs and about 2700 base pairs were found. This means that this plasmid is compatible with the SmaI cleavage site of pUC13 and Eco
This proves to be a recombinant plasmid in which a fragment A consisting of about 400 base pairs was inserted between the RI cleavage sites. Hereinafter, this recombinant plasmid is abbreviated as PAM11. 50 μg of plasmid PAM11 DNA was cleaved with 10 units of restriction endonucleases BamHI and 10 units of EcoRI to give approximately 40
A DNA fragment consisting of 0 base pairs (hereinafter abbreviated as fragment B) was obtained.

This fragment B contains the entire DNA base sequence of the first half of the α-amylase gene lacking the region involved in gene expression and secretion of the resulting protein.

Next, 50 μg of DNA containing the α-amylase gene derived from Bacillus subtilis (described above) was digested with restriction endonucleases EcoRI and pvuII, and the latter half of the α-amylase gene consisting of approximately 1300 base pairs was DNA.
A fragment (hereinafter referred to as fragment C) was prepared.

In addition, the restriction endonucleases BamHI and HincII
Fragment B and fragment C were ligated with the plasmid pUC12 DNA (Pharmacia), which had been digested with (Takara Shuzo) and then treated with E. coli alkaline phosphatase.

For the reaction, 5 μg of plasmid pUC12 DNA was used and HincII
10 units BamHI 10 units were performed 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 a final concentration of 0.1 M, and the temperature was adjusted to 65 ° C2.
Allowed to react for hours.

After the reaction was completed, the reaction solution was subjected to phenol extraction, ether extraction, and ethanol precipitation to collect DNA. After that, 2
0μl of 50 m M Tris - was redissolved in HCl buffer.

1 μg of the pUC12 DNA thus obtained and 2 μg of fragment B and 2 μg of fragment C dissolved in Tris-HCl buffer (pH 7.5)
g was ligated with E. coli T ₄ ligase (Takara Shuzo) in the same manner as described above.

E. coli K-12 (JM10
1) The strain was transformed to obtain a transformant showing ampicillin resistance. Plasmid DNA was prepared from the obtained ampicillin-resistant strain by the alkali method.

Restriction endonuclease Eco
When treated with RI alone and with restriction endonucleases BamHI and HindIII, a DNA fragment of about 4500 base pairs, which was cleaved at one site by EcoRI treatment,
About 1 when processed with amHI and HindIII (Takara Shuzo)
DNA fragments of 800 base pairs and about 2700 base pairs were confirmed, respectively.

Combining the above results, pUC1 used as a vector
The plasmid in which 2 was about 2700 base pairs (hereinafter, abbreviated as pAM29) was used as a BamHI cleavage site of pUC12 and H.
It was confirmed to be a recombinant plasmid in which the α-amylase gene lacking the region involved in gene expression and the secretion of the resulting protein was inserted between the indIII cleavage sites.

The ligation site between the region containing the α-amylase gene of this plasmid pAM29 and pUC12 is shown in FIG. p
The restriction endonuclease BamH was added upstream from the amino terminus of the α-amylase gene portion of AM29 plasmid DNA.
There are respective cleavage sites of I, SmaI, and SstI. In addition, HindIII and PvuII cleavage sites are present downstream from the carboxyl terminus, respectively.

2) Binding of pES150 to α-amylase gene and secretory production of α-amylase 1 μg of pAM29 obtained in 1) was cleaved with restriction endonucleases SstI and HindIII according to a conventional method, and then α
The 1.8 kb fragment containing the amylase gene was separated and collected by low melting point agarose gel electrophoresis and purified by phenol extraction, ether extraction and ethanol precipitation.

The purified DNA was dried, dissolved in 32 μl of distilled water, and the protruding end portion was removed using T ₄ DNA polymerase under the same conditions as in Example 1 to make a blunt end. The same method as in Example 1 was used for purification after the reaction with T ₄ DNA polymerase.

The purified DNA is dried 20μl of 50 m M Tris - with out 10μl was dissolved in HCl buffer (pH 7.5), likewise smooth as in Example 1 by cutting after ring opening T ₄ polymerase with restriction endonuclease SphI Terminated pES150 (0.5 μg) and T ₄
Ligated by ligase. The binding conditions are the same as those used in Example 1.

The resulting reaction mixture was used to transform α-amylase-deficient Bacillus subtilis 1A289 strain (Ohio University Bacillus stock center preserved strain) by the protoplast method, and then 1% soluble starch and 150 μg / kanamycin in DM-3 regeneration medium were used. Plated in ml. After culturing at 37 ° C. for 24 hours, a plate was sprayed with an iodine solution to obtain 50 transformants that form halo. A plasmid was prepared from the MT-33 strain, which is one of these, and a physical map was created using various restriction endonucleases, and it was found that the downstream of the DNA base sequence encoding the pro-structure of the extracellular neutral protease of pES150. It was found that the DNA base sequence was bound to the DNA base sequence containing the region encoding the α-amylase gene of about 1.8 kb (pPA33 and command).

pPA33 has the DNA base sequence shown in FIG. 1 in a form in which it is connected to the upstream of the α-amylase gene from the DNA base sequence encoding the prostructure cleavage point of the extracellular neutral protease. Restriction endonucleases SmaI and BamHI cleavage sites are present in this DNA base sequence.

Bacillus subtilis MT-33 containing this recombinant DNA pPA33 was treated with BY medium (H. Uehara et al. J. Bacteriol. 1974, 119 ,
82) and incubated at 37 ° C for 18 hours, and
-The amylase activity was determined by the iodine-starch method (H. Fuwa J. Bioch.
As a result of measurement using em (Tokyo) 1954, 41 , 583), it was confirmed that 2500 units of the enzyme was secreted per 1 ml of the culture medium supernatant. The activity of the 1A289 strain used as a control was below the detection limit.

Example 3 Secretory production of human interferon-β Human interferon-β (hIFN-β) was prepared using plasmid pIF20 containing the gene. The plasmid was constructed using synthetic DNA so that the restriction endonuclease Sau3AI cleavage sites were present at both ends of the DNA base sequence encoding the mature interferon in which the signal portion involved in the secretion of hIFN-β was deleted, When the plasmid is digested with Sau3AI, a 0.5 kb DNA base sequence encoding the mature protein of hIFN (hereinafter referred to as hIFN structural gene) is obtained.

1 μg of the hIFN structural gene was added to pPA3 constructed in Example 2.
3 (1 μg) was inserted and ligated to the BamHI cleavage site using T ₄ ligase. The binding conditions are the same as in Example 1.

Using this reaction mixture, Bacillus subtilis strain 1A510 was transformed by the protoplast method, and 20 strains of colonies that did not form a large halo on DM-3-kanamycin (150 μg / ml) medium containing 1% starch were selected. Selected.

The transformant is used in BY medium (kanamycin 5 μg / ml added)
At 30 ° C. for 18 hours, the culture supernatant was used to measure hIFN-β activity by the EIA method. As a result, it was confirmed that the transformant ^# 25 secreted 5 × 10 ⁴ U / ml IFN into the culture supernatant.

As a result of preparing and examining a plasmid from the strain, it was confirmed to be the one shown in FIG. 6 (named pPIF25).

^{The #} 25 transformant was prepared with 2.5 times the concentration of BY medium (kanamycin 5
[mu] g / ml containing) when 30 ° C. 18 hours at plus 10 m M PMSF, IFN activity in the supernatant reached ⁵ × 10 5 U / ml.

[Brief description of drawings]

The respective drawings will be briefly described as follows. FIG. 1 is a diagram showing a DNA base sequence having cleavage sites for restriction endonucleases SmaI and BamHI. -: Restriction endonuclease recognition site, ▲: restriction endonuclease cleavage site. FIG. 2 is a diagram showing a construction process from pNP150 to pES150, pSB150 and pBS150. P: promoter, PP: DNA base sequence encoding pre-pro structure, Mat: DNA base sequence encoding mature protein. FIG. 3 is a view showing the DNA base sequence of the ligation site between pUC13 and fragment A. The inside is the DNA base sequence of fragment A. FIG. 4 is a diagram showing the DNA base sequence of the ligation site between the region containing the α-amylase gene of pAM29 and pUC12. Inside is the DNA base sequence of the region containing the α-amylase gene. Fig. 5 shows the construction process and physical map of pPA33. Fig. 6 is a diagram showing a physical map of pPIF25. Fig. 7 DNA sequence involved in expression of extracellular neutral protease gene of Bacillus amyloliquefaciens, DNA sequence encoding pre-pro structure and partial mature protein of extracellular neutral protease It is a figure which shows a DNA base sequence. In the figure, the first to 251st DNA base sequences counted from the 5'end upstream of the DNA base sequence are DNA base sequences involved in the expression of the extracellular neutral protease gene.
The 914th to 914th nucleotide sequences are DNA nucleotide sequences encoding the pre-pro structure, and the 915th and subsequent nucleotide sequences are DNA nucleotide sequences encoding a part of the mature protein. In addition, A, C, G, and T in the figure of the DNA base sequence of the present invention represent adenine, cytosine, guanine, and thymine, respectively.

Continuation of front page Examiner Yuko Saeki (56) References European Patent Publication 133756 (EP, A) J. Bacteriol. , 159 (3) (1984) P. 811-819 J.I. Biotech. , 1 (1984) p. 265-278

Claims (9)

(57) [Claims] 1. An expression / secretion vector having a DNA base sequence involved in the expression of an extracellular neutral protease gene of Bacillus amyloliquefaciens and a DNA base sequence encoding a pre-pro structure, which further comprises: -An expression / secretion vector characterized in that a heterologous gene is bound immediately after the DNA base sequence encoding the pro structure. 2. D encoding a DNA base sequence and a pre-pro structure involved in the expression of extracellular neutral protease gene.
The expression / secretion vector according to claim 1, characterized in that the NA base sequence contains a part of the bases shown below. 3. An extracellular neutral protease of Bacillus amyloliquefaciens is encoded near the cleavage point of the pro-structure.
The restriction endonuclease SphI cleavage site present in the DNA base sequence is cleaved, the resulting protruding end is made blunt by an enzyme having exonuclease activity, and then the DNA base sequence containing the region encoding the desired protein is ligated. The expression according to claim 1, characterized in that a DNA base sequence having a restriction ennuclease cleavage site that can be used in the case is constructed by binding to the blunt end.
Secretion vector. 4. A DNA base sequence having a restriction endonuclease cleavage site that can be used when binding a DNA base sequence containing a region encoding a desired protein, including the following: The expression / secretion vector according to item 3 above. 5'CGCCCGGGGATCCCC3 '3'GCGGGCCCCTAGGGG5' 5. Encoding in the vicinity of the prostruction cleavage point of the extracellular neutral protease of Bacillus amyloliquefaciens
The restriction endonuclease SphI cleavage site present in the DNA base sequence is cleaved, the resulting protruding end is made blunt by an enzyme having exonuclease activity, and then the DNA base sequence containing the region encoding the desired protein is ligated. When used, the α-amylase gene of Bacillus subtilis from which the DNA base sequence encoding the secretory signal region has been removed via the DNA base sequence having a restriction endonuclease cleavage site as shown below is bound. The expression / secretion vector according to claim 1, characterized in that 5'CGCCCGGGGATCCCC3 '3'GCGGGCCCCTAGGGG5' 6. The expression / secretion according to claim 1, wherein the part involved in replication of the expression / secretion vector is derived from a plasmid or a phage capable of replication in a bacterium of the genus Bacillus. vector. 7. A Bacillus when binding a DNA base sequence containing a region encoding a desired protein to an expression / secretion vector.
The method according to claim 1, characterized in that the binding is carried out by using a restriction endonuclease cleavage site present in a DNA base sequence encoding the vicinity of the pro-structure cleavage point of the extracellular neutral protease of amyloliquefaciens. Expression / secretion vector 8. The expression / secretion vector according to claim 7, wherein the restriction endonuclease cleavage site is SphI. 9. The expression / secretion vector according to claim 5, wherein the desired protein is human β-interferon.