GB2171703A - Expression-secretion vector - Google Patents

Expression-secretion vector Download PDF

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GB2171703A
GB2171703A GB08601033A GB8601033A GB2171703A GB 2171703 A GB2171703 A GB 2171703A GB 08601033 A GB08601033 A GB 08601033A GB 8601033 A GB8601033 A GB 8601033A GB 2171703 A GB2171703 A GB 2171703A
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dna sequence
expression
secretion
gene
protein
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GB8601033D0 (en
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Yoshio Furutani
Masaru Honjo
Akira Nakayama
Hiroaki Shimada
Izumi Mita
Toshinori Ozaki
Kazuaki Manabe
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National Institute of Advanced Industrial Science and Technology AIST
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Priority claimed from JP60005834A external-priority patent/JPS61166400A/en
Priority claimed from JP60053433A external-priority patent/JP2500311B2/en
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

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Abstract

A host bacterium of the genus Bacillus is transformed with recombinant DNA formed by joining or inserting a DNA sequence containing a gene coding for a desired protein into a vector containing a gene coding for an extracellular enzyme, in particular, neutral protease which is naturally produced and secreted in large amounts by bacteria of the genus Bacillus, and is then cultured. The desired protein can be efficiently produced and secreted by the host. The desired protein can be easily isolated from the culture medium. <IMAGE>

Description

SPECIFICATION Expression-secretion vector for gene expression and protein secretion, recombinant DNA including the vector, and method of producing proteins by use of the recombinant DNA Background of the invention 1. Field of the invention This invention relates to an expression-secretion vector involved in the expression of a gene and the secretion of the protein thus produced, a recombinant DNA formed by joining a DNA sequence containing a gene coding for a desired protein to such a vector, and a method of producing proteins by using such a recombinant DNA.
More particularly, the present invention relates to a method of producing proteins which comprises preparing an expression-secretion vector having a DNA sequence containing a region involved in the expression of a gene coding for an extracellular enzyme, which is produced and secreted in large amounts by a bacterium of the genus Bacillus, and the secretion of the protein thus produced, forming a recombinant DNA by joining a DNA sequence containing a gene coding for a desired protein to the expression-secretion vector, introducing the recombinant DNA into a bacterium of the genus Bacillus used as the host, and thereby causing the bacterium to produce and secrete the desired protein.
2. Description of the prior art As DNA sequences involved in the expression of a gene, the promoter region containing the -35 and -10 regions which are the sites for recognition and binding of RNA polymerase, and the ribosome binding region through which the messenger RNA synthesized by RNA polymerase binds ribosomes are known to be important. In addition to the DNA sequences of these regions, the number of nucleotides present between these regions and the number of nucleotides present between the ribosome binding region and the translation initiation codon are also known to be important.
Moreover, in order to secrete the protein produced as a result of gene expression into the culture medium, the polypeptide, called "a secretion signal", located upstream from the mature protein is essential.
It has been demonstrated that this polypeptide is synthesized within the cells in the form joined to the upstream side of the amino end of the mature protein and, during the secretion of the mature protein, plays an important role in its passage through the cell membrane. Accordingly, the region coding for the aforesaid polypeptide is essential to the secretion of the protein produced as a result of gene expression.
For example, neutral protease, which is an extracellular enzyme produced by microorganisms, is synthesized within the cells in the form of a precursor having a polypeptide as described above, called "a signal sequence", as a region necessary for the extra-cellular secretion of the enzyme.
This polypeptide is joined to the N-terminus of a polypeptide (pro-peptide) located upstream of the Nterminus of the mature neutral protease to be secreted into the medium and removed during the process of secretion, i.e., joined to the N-terminus of pro-neutral protease. Thus, this peptide is known to play an important role in the passage of the enzyme through the cell membrane during the process of secretion [G. Blobel, J. Cell Biol., 67, 835(1975)].
The aforesaid signal sequence and pro-peptide are present in the form a single polypeptide called "prepro-peptide". Since the extracellularly secreted mature neutral protease does not have the prepropeptide portion, this portion is considered to be successively split off during the process of secretion. It may be readily understood that this splitting process is related to the efficient secretion of neutral protease. Accordingly, the DNA sequence coding for the prepro-peptide is an important region for the secretion of neutral protease produced as a result of gene expression.
As can be seen from the above description, expression-secretion vectors having a DNA sequence involved in the expression of a gene and a DNA sequence coding for a prepro-peptide have an important significance for the purpose of effecting the expression of a foreign gene and the extracellular secretion of the protein thus produced.
The marked development of genetic engineering in recent years has made it possible to produce foreign proteins by the use of microorganisms. However, in Escherichia coli commonly used as the host, the pro-duction of a desired protein has a certain limit because it is accumulated within the cells. Moreover, it is very difficult to separate the desired protein from other intracellular proteins through the process of purification.
Furthermore, the desired protein, though purified, may often be contaminated with pyrogens originating from Escherichia coli. In these respects, the production of materials by genetic engineering techniques using Escherichia coli as the host involves serious problems from a practical point of view.
On the other hand, since bacteria of the genus Bacillus lack pathogenicity and have long been used in the production of antibiotics, extracellular enzymes, nucleotide seasonings and the like by extracellular secretion, it would be of great industrial significance from the viewpoint of microbial production of materials to create an expression-secretion vector suitable for use in such bacteria.
Summary of the invention It is an object of the present invention to provide an expression-secretion vector permitting the expres sion of a gene coding for a desired protein in a host microorganism and the extracellular secretion of the protein produced within the host cells as a result of the gene expression and, in particular, an expression-secretion vector permitting high-level expression of a gene in a bacterium of the genus Bacillus and efficient extracellular secretion of the protein thus produced.
It is another object of the present invention to provide a recombinant DNA formed by joining a DNA sequence containing a gene coding for a desired protein to such an expression-secretion vector and an efficient method of producing desired proteins by using such a recombinant DNA according to genetic engineering techniques.
These objects of the present invention can be accomplished by an expression-secretion vector comprising a vector portion containing a region involved in its replication in host microorganisms, and a DNA sequence containing a region involved in the expression of a gene coding for an extracellular protein (in particular, the extracellular neutral protease of a bacterium of the genus Bacillus) and the secretion of the protein thus produced; a recombinant DNA formed by joining a DNA sequence containing a gene coding for a desired foreign protein to the expression-secretion vector; and a method of producing proteins which comprises transforming a host micro-organism with the recombinant DNA, culturing the resulting transformed strain, and then recovering the desired protein from the culture medium.
Brief description of the drawings Figure 7 illustrates a DNA sequence having cleavage sites for the restriction endonucleases Smal and BamHI, the solid lines (-) representing the restriction endonuclease recognition sites and the closed triangles (A) representing the restriction endonuclease cleavage sites; Figure 2 illustrates the DNA sequence of the junction between pUC13 and fragment A as described in Example 3, the enclosed portion representing the DNA sequence of fragment A; Figure 3 illustrates DNA sequence of the junction between pUC12 and the a-amylase gene-containing region of pAM29 as described in Example 3, the enclosed portion representing the DNA sequence of the a-amylase gene-containing region; Figure 4 illustrates the procedure for creating Bacillus subtilis strain OZ361 as described in Example 2;; Figure 5 illustrate the DNA sequence of one strand of the junction between human interferon-, DNA and plasmid pNPi50 in the plasmid pOZ361 obtained in Example 2; Figures 6, 7, 8, and 9 illustrate the procedures for creating plasmids pNPA74, pNP174, pBS150 & BR< pSB150, and pPA33, respectively; and Figure 10 is a physical map of plasmid pPIF25.
In these figures, A, C, G and T represent adenine, cytosine, guanine and thymine, respectively. In addition, P represents a promoter region, P-P represents a region containing a DNA sequence coding for the prepro-peptide, and Mat. represents a region containing the structure gene for a mature protein.
Detailed description of the prepared embodiments The expression-secretion vector of the present invention comprises a DNA sequence containing a region involved in the expression of a gene and the secretion of the protein thus produced, and a vector portion containing a region involved in its replication in host microorganisms. By joining a DNA sequence containing a gene coding for a desired protein to the aforesaid expression-secretion vector, it become possible to effect the production and extracelluiar secretion of the desired protein.More specifically, this can be accomplished by joining a DNA sequence containing a gene coding for a desired protein to the aforesaid expression-secretion vector on the downstream side of the DNA sequence containing the region involved in the expression of a gene and the secretion of the protein thus produced; introducing the resulting recombinant DNA into a host microorganism; culturing the host microorganism so as to cause the gene coding for the desired protein to be expressed in the host microorganism and also cause the protein thus produced to be secreted in large amounts, for example, into the culture medium of the host microorganism; and then recovering the protein from the supernatant of the culture medium and purifying it according to a simple procedure.
The vector portion possessed by the expression-secretion vector of the present invention may be obtained from a member selected, according to the need, from the group consisting of plasmids, phages and derivatives thereof that are capable of replication in the host microorganism into which a recombinant DNA formed with the expression-secretion vector will be introduced.
Where a bacterium of the genus Bacillus is used as the host for the previously described reasons, the vector portion may comprise any vector that are capable of replication in Bacillus bacteria. However, plasmids having a gene defining resistance to an antibiotic are convenient for the purpose of selecting transformed cells after the introduction of a recombinant DNA.
Among such antibiotic-resistant plasmids, pUB110, pTP5, pUC194, pE194, pSA0501, pBD6 and derivatives thereof are being commonly used and generally available.
Among phages which are capable of replication in Bacillus bacteria, per15, 29, 105, pll and the like are generally available.
Basically, the expression-secretion vector of the present invention comprises a vector portion as defined above and a DNA sequence joined to the vector portion and containing a region directly involved in the expression of a gene coding for a protein and the secretion of the protein thus produced (which will hereinafter referred to as the expression-and secretion-related DNA sequence).
As the expression- and secretion-related DNA sequence, there may be used DNA sequences, which is isolated, for example, from the chromosomes, plasmids and the like of microorganisms having the property of secreting a protein extracellularly, and which contains a region involved in the expression of the gene coding for the extracellular protein and the extracellular secretion of the protein thus produced within the cells.
Such expression- and secretion-related DNA sequences can be obtained, for example, by isolating chromosomes from a microorganism secreting a protein extracellularly in the usual manner, cleaving the chromosomes with a suitable restriction endonuclease to obtain a DNA sequence containing a region involved in the expression of the extracellular protein gene and the secretion of the protein thus produced, and then hydrolyzing the phosphodiester bonds of the DNA sequence (or digesting the DNA sequence) from its downstream end to delete an unnecessary part in such a way that the DNA sequence containing the region involved in the expression of the gene and the secretion of the protein thus produced is retained.The step of digesting the DNA sequence in such a way that a DNA sequence containing the region involved in the expression of the gene and the secretion of the protein thus produced is retained may be carried out by the use of any suitable nuclease. However, it is convenient to use exonuclease Ba131 for this purpose.
Where a bacterium of the genus Bacillus is used as the host for the previously described reasons and the gene coding for the desired protein is expressed by using the expression-secretion vector of the present invention in the Bacillus bacterium, the region, which is contained in the aforesaid expression- and secretion-related DNA sequence of the expression-secretion vector of the present invention, and which is involved in the expression of a gene and the secretion of the protein thus produced, should preferable by one originating from a bacterium of the genus Bacillus [Goldfarb, D.S., et al., Nature, 293, 309(1981)], because the RNA polymerases and ribosomes of Bacillus bacteria have strict sepcificity in the recognition of the promoter region and the ribosome binding region [Sueharu Horinouchi, Tanpakushitsu-Kakusan Koso, 28, 1468(1983)].
Moreover, it is desirable from a practical point of view that, for purposes of high-level expression and efficient secretion, the above region, which is contained in the expression-secretion related DNA sequence, and which is involved in the expression of a gene coding for a protein and the secretion of the protein thus produced, should be one derived from a gene coding for an extracellular enzyme which is produced and extracellularly secreted in large amounts within a short period of time. Well-known examples of such extracellular enzymes originating from Bacillus bacteria include neutral protease, alkaline protease, a-amylase, levansucrase and the like. Any of the genes coding for these enzymes can be used for the purpose of constructing the expression-secretion vector of the present invention.Among them, the neutral protease of Bacillus amyloliquefaciens is a protein produced and secreted in large amounts within a shorter period of time. For this reason, the region, which is obtained from the neutral protease gene of Bacillus amylolique-faciens and, which is involved in the expression of the gene and the secretion of the protein thus produced, i.e., the region having the DNA sequence involved in the expression of the neutral protease gene and the DNA sequence coding for the prepro-peptide, is especially suitable for the formation of the expression-and secretion-related DNA sequence possessed by the expression-secretion vector of the present invention.
As the DNA sequence involved in the expression of the neutral protease gene and the DNA sequence coding for the prepro-peptide, both derived from the extracellular neutral protease gene of Bacillus amyloliquefaciens, those derived from the neutral protease gene, whose entire DNA sequence has already been determined by the present inventors (as described in Japanese Patent Application No. 175158/'84, (United States Patent Application Serial No. 686, 892 and EP-149 241-A-2) and set forth in claim 7, are convenient for use in the expression-secretion vector of the present invention, because their structures have been elucidated.
Using the aforesaid expression-secretion vector of the present invention having the vector portion capable of replication in host microorganisms and the expression- and secretion-related DNA sequence, a recombinant DNA, which is capable of replication in host microorganisms and permits the expression of a gene coding for a desired protein and the extracellular secretion of the desired protein thus produced, can be formed by inserting a DNA sequence containing the gene coding for the desired protein into the expression-secretion vector downstream from its region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced.
Accordingly, the expression-secretion vector of the present invention must have a site at which a DNA sequence containing a gene coding for a desired protein can be joined thereto on the downstream side of the expression- and secretion-related DNA sequence and downstream from its region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced.
The expression-secretion vector meeting this requirement can be obtained, for example, by using an expression- and secretion-related DNA sequence having a terminus and/or one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be joined thereto or inserted thereinto downstream from its region involved in the expression of a gene coding for a protein and secretion of the protein thus produced.
Using the expression-secretion vector thus obtained, a DNA sequence containing a gene coding for a desired protein can be, either directly or with the aid of suitable linkers, joined thereto or inserted thereinto at the aforesaid terminus or one of the aforesaid restriction endonuclease cleavage sites. Alternatively, this may be done after the aforesaid terminus or one of the aforesaid restriction endonuclease cleavage sites has been suitably digested, for example, with an exonuclease.
Especially in the case of the extracellular neutral protease gene of Bacillus amyloliquefaciens, the DNA sequence coding for the cleavage site of propeptide has a restriction site for the restriction endonuclease Sphl. Accordingly, if an expression- and secretion-related DNA sequence derived from this neutral protease gene is used, a DNA sequence containing a gene coding for a desired protein can be joined thereto at that Sphl cleavage site.
That is, a recombinant DNA necessary for the production and secretion of a desired protein can be constructed by joining a DNA sequence containing the gene coding for the desired protein to the aforesaid Sphl cleavage site either directly or indirectly with the aid of linkers or the like.
Thus, when an expression- and secretion-related DNA sequence derived from the neutral protease gene is used as the expression-secretion vector of the present invention, it includes the DNA sequence involved in the expression of the neutral protease gene and the DNA sequence coding for the prepropeptide of neutral protease, as described before, so that the expression of a gene coding for a desired protein is effected by the action of the former DNA sequence and the same polypeptide as the prepropeptide of neutral protease is added to the N-terminus of the desired protein by the action of the latter DNA sequence.
The presence of the aforesaid polypeptide permits the desired protein to be secreted out of the cells of the Bacillus bacterium used as the host.
Under the category of the expression-secretion vector of the present invention are also included vectors formed by joining a DNA sequence having a terminus and/or one or more restriction endonuclease cleavage sites suitable for joining a DNA sequence containing a gene coding for a desired protein, to the above-defined expression-secretion vector on the downstream side of the expression- and secretion- related DNA sequence containing the region involved in the expression of a gene and the secretion of the protein thus produced, so that a DNA sequence containing a gene coding for a desired protein may be readily joined thereto either directly or with the aid of suitable linkers. For this purpose, the present inventors created a DNA sequence containing the nucleotide sequence shown in Figure 1.
This DNA sequence has cleavage sites for the restriction endonucleases Smal and BamHI and, therefore, is convenient for joining a DNA sequence containing a gene coding for a desired protein to the expression-secretion vector of the present invention.
The expression-secretion vector of the present invention having a DNA sequence containing the nucleotide sequence shown in Figure 1 and an expression-and secretion-relatbd DNA base sequence derived from the neutral protease gene of Bacillus amyloliquefaciens can be prepared, for example, from a DNA sequence prepared by cleaving the neutral protease gene of Bacillus amyloliquefaciens at the Sphl cleavage site located in the DNA sequence coding for the cleavage site of the pro-peptide of the neutral protease, treating the cleaved gene with an enzyme having exonuclease activity to remove the singlestranded terminius located on the downstream side of the DNA sequence coding for the prepro-peptide and thereby generate flush end, and then joining thereto a DNA sequence containing the nucleotide sequence shown in Figure 1.
The expression-secretion vector of the present invention may have a gene coding for a readily distinguishable extracellular enzyme, downstream from its region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced.
For example, the vector (Figure 9) which is formed by cleaving the neutral protease gene at the Sphl cleavage site located in the DNA sequence coding for the cleavage site of pro-peptide, treating the cleaved gene so as to generate flush end, and then joining thereto a DNA sequence contains the nucleotide sequence shown in Figure 1 and a DNA sequence joined to its downstream end and containing the amylase gene of a Bacillus bacterium (but deprived of the DNA sequence coding for its own secretion signal), is also included under the category of the expression-secretion of the present invention.
In this expression-secretion vector, the DNA sequence containing the nucleotide sequence shown in Figure 1 and a DNA sequence joined thereto and containing the a-amylase gene is located immediately downstream from the DNA sequence containing a region involved in the expression of the neutral protease gene and the DNA sequence coding for its prepro-peptide.
When this recombinant DNA is introduced into a bacterium of the genus Bacillus which does not produce or secrete amylase, the a-amylase gene is expressed by the action of the DNA sequence involved in the expression of the neutral protease gene, so that prepro-a-amylase having the prepro-peptide of neu tral protease added to the upstream side of the N-terminus is synthesized within the cells.
By the action of this prepro-peptide, the resulting product is secreted into the culture medium. However, the prepro-peptide is split off during the process of secretion and mature a-amylase is accumulated in the culture medium.
Where the desired protein is a-amylase, this expression-secretion vector constitutes a recombinant DNA useful for the production of amylase.
In this recombinant DNA, the nucleotide sequence shown in Figure 1 is present between the amylase gene and the flush-ended Sphl cleavage site located in the DNA sequence coding for the cleavage site of the pro-peptide. Accordingly, by utilizing the Smal and BamHI cleavage sites present in that necleotide sequence, a DNA sequence containing a gene coding for a new desired protein can be joined to this recombinant DNA.
Thus, this recombinant DNA may also be said to be an expression-secretion vector. Specifically, where this expression-secretion is used as such, the host microorganism produces a-amylase. However, once a DNA sequence containing a gene coding for a desired protein is inserted into the expression-secretion vector at the aforesaid Smal or BamHI cleavage site, the production and secretion of a-amylase by the action of the amylase gene present in the expression-secretion vector is stopped.
Accordingly, where a strain which does not produce or secrete os-amylase is used as the host, the step of selecting transformed cells having a recombinant DNA formed by joining a DNA sequence containing a gene coding for a desired protein to the expression-secretion vector can be markedly simplified.
More specifically, it may be understood that, when cultured on a starch-containing agar medium, bacterial cells forming a halo by the starch-iodine reaction have the expression-secretion vector per se, while bacterial cells forming no halo have a recombinant DNA formed by inserting a DNA sequence containing a gene coding for a desired protein into the expression-secretion vector.
The expression-secretion vector of the present invention having the above-described construction can be formed, for example, by joining a vector DNA containing a region involved in its replication in host microorganisms to an expression- and secretion-related DNA sequence. Alternatively, the expression-secretion vector can also be formed by joining an extracellular protein gene having a portion constituting an expression- and secretion-related DNA sequence to a vector DNA capable of replication in host microorganisms so as to form a plasmid, and deleting at least the structural gene for the extracellular protein so as not to impair the functions of the desired expression-secretion vector.
Using the expression-secretion vector of the present invention, a recombinant DNA useful for the production and secretion of a desired protein in a host microorganism can be formed by joining thereto or inserting thereinto a DNA sequence containing the gene coding for the desired protein, either directly or with-the aid of suitable linkers, at the terminus or one of the restriction endonuclease cleavage sites located downstream from the region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced.
The DNA sequence containing the gene coding for the desired protein, which may be joined to the expression-secretion vector of the present invention, comprises a DNA sequence containing the structure gene defining the structure of the desired protein and a terminator region. Where the desired protein is inherently secreted out of the cells, the secretion signal region is removed from the protein gene as the gene coding for the desired protein.
Using the expression-secretion vector of the present invention, there may be obtained recombinant DNAs useful for the production of such proteins as interferon, growth hormone, interleukin, nerve growth factor, kallikrein, plasminogen activator, and other physiologically active polypeptides or enzymes. In order to form these recombinant DNAs, a DNA sequence containing the gene coding for the desired protein may be joined to or inserted into the expression-secretion vector of the present invention either directly or with the aid of suitable linkers.
Furthermore, using the recombinant DNA so formed, the desired protein coded for by the gene incorporated into the recombinant DNA may be produced and secreted in large amounts. This can be accomplished by transforming a host microorganism with the recombinant DNA according to genetic engineering techniques and then culturing the resulting transformed strain.
A variety of microorganisms may be used as the host for the production of the protein. However, as previously described, bacteria of the genus Bacillus are convenient because they lack pathogenicity, have long been used in the production of antibiotics, extracellular enzymes, nucleotide seasonings and the like, and have well-defined properties.
As the host for the recombinant DNA of the present invention, there may be used any of various Bacillus bacteria including Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus megaterium Bacillus stearothermophilus and the like. Among them, Bacillus subtilis which has been very closely studied in the field of genetics is easy to use.
Transformation of the host microorganism may be carried out according to any method. However, where Bacillus subtilis is used as the host, the protoplast method [S. Chang and S.N. Cohen, Mol. Gen.
Genet., 168, 111(1979)] and the competent cell method [C. Anagnostopoulos and J. Spizizen, J. Bacteriol., 81, 741(1961)] are effective.
The desired protein produced and secreted by the transformed strain can be readily recovered, for example, by filtering or centrifuging the culture medium to remove the cells therefrom, and then isolating the desired protein from the filtrate or supernantant according to any conventional procedure for the purification of extracellular enzymes and the like.
Thus, as compared with the case in which the desired protein is accumulated within the cells, for example, of Escherichia coli, the method of the present invention permits marked simplification of the purification process and, therefore, is particularly advantageous from the viewpoint of industrial production of materials.
The present invention is further illustrated by the following examples. However, these examples are not to be construed to limit the scope of the present invention.
Example I (Preparation of a plasmid containing the neutral protease gene of Bacillus amyloliquefaciens) Bacillus amyloliquefaciens strain F (Deposition No. ATCC 23350; a stock strain maintained in the American Type Culture Collection, 12301, Parklawn Drive 19, Rockville, Maryland 20852-1776) was cultured in 2 liters of a broth medium (Difco Nutrient Broth) at 37"C for 15 hours. After the cells were collected, chromosomal DNA was prepared according to the method of Saito and Miura [Saito, H., and Miura, K-l., Biochem. Biophys. Acta, 72, 619(1963)], and then 10 mg of purified chromosomal DNA was obtained.
Using 100 units of the restriction endonuclease Sau3Al (Takara Shuzo Co.), 500 ig of this chromosomal DNA was digested at 37"C for 5 minutes. In addition to Sau3Al and DNA, the reaction system contained 7mM MgCl2 and 100mM NaCI in a 10mM Tris-HCI buffer solution (pH 7.5). After completion of the reaction, 1 ll9 of the DNA was analyzed by 1% agarose gel electrophresis. This revealed that the reaction product was a partial degradation product of the donor chromosomes composed mainly of DNA fragments having a size of 2-8 kb. Then, the remainder of the reaction product was subjected to electrophoresis in 0.7% low-melting agarose gel at 100V for 3 hours.After the gel portion corresponding to a 1.5-9 kb fraction was cut out, DNA was purified by extraction with phenol and with chloroform, and then recovered by precipitation with ethanol. The recovered DNA was dissolved in 200 ,wl of 50mM Tris-HCl buffer solution (pH 7.5) and used as donor chromosomal DNA fragments in the following experiment.
The donor chromosomal DNA fragments thus obtained were inserted into the BamHI restriction site of the plasmid pUB110, which had been treated with the Escherichia coli alkaline phosphatase (Worthington Co.) to hydrolyze the terminal phosphate after complete cleavage with endonuclease BamHI (Takara Shuzo Co.).
More concretely, the treatment of pUB110 with BamHI was carried out by incubating the reaction system at 37"C for 4 hours. The reaction system contained 100 ,ug of pUB110, 50 units of BamHI (Takara Shuzo Co.), 7mM MgCl2, 100mM NaCI, 2mM 2-mercapto-ethanol and 0.01% bovine serum albumin in a 10mM Tris-HCI buffer solution (pH 8.0). The resulting BamHI-cleaved pUB110 was extracted with phenol three times and then recovered by precipitation with ethanol. The recovered pUB110 was treated with the alkaline phosphatase of Escherichia coli by dissolving it in a 0.1M Tris-HCI buffer solution (pH 8.0) containing 5 units of BAPF (Worthington Co.) and incubating this reaction system at 65"C for 4 hours.Thereafter, the resulting pUB110 was extracted with phenol and then recovered by precipitation with ethanol.
The recovered pUB110 was dissolved in 100 Fl of a 50mM Tris-HCI buffer solution (pH 7.5).
Using T4 ligase (Takara Shuzo Co.), the donor chromosomal DNA fragments previously obtained were combined with the BamHI- and phosphatase-treated pUB110. The reaction system contained 50 Al of the donor chromosomal DNA fragments, 20 pbl of pUB110, 5 units ofT4 ligase, 6.6mM MgCl2, 10mM dithiothreitol and 1 mM ATP (adenosine triphosphate) in a 66mM Tris-HCI buffer solution (pH 7.5). The reaction was carried out at 150C for 4 hours. After completion of the reaction, a sample was analyzed by 1% agarose gel electrophoresis. This revealed that pUB110 used as the vector was joined to the donor chromosomal DNA segments to form recombinant DNA molecules.
Using the recombinant DNA molecules thus obtained, transformation of Bacillus subtilis was carried out according to the protoplast method of Chang [Chang, S., and Cohen, S.N., Mol. Gen. Genet., 168, 111(1978)]. The protoplastswere regenerated in a medium containing kanamycin sulfate at a final concentration of 100 FLg/ml. As the host for cloning, Bacillus subtilis strain 1A274 (a stock strain maintained in the Bacillus Genetic Stock Center, the Ohio State University, 484 West, 12th Avenue, Columbus, Ohio 43210, U.S.A.) was used.
The kanamycin-resistant colonies obtained by the transformation were transferred onto the TBAB agar media (Difco) containing 0.8% casein and 40 Fg/ml kanamycin, incubated at 37"C for 14 hours and then examined for the presence of a halo around each colony. Among about ten thousand kanamycin-resistant colonies tested, one strain (+150) formed a significantly large halo around the colony.
The large halo forming strain obtained by isolating the single colony of the above-described transformed strain #150 was cultured in 50 ml of the Penassay medium (Difco) at 370C for 14 hours. Thereafter, the cells were collected, washed with a 50mM Tris-HCI buffer solution (pH 7.5) containing 5mM EDTA and 50mM NaCI, and then used in the preparation of a plasmid by the alkaline method [Birnboim, H.C., and Poly, J., Nucleic Acid Pews., 7, 1513(1979)]. The resulting plasmid was treated with the restriction endonuclease EcoRI, Bg1ll and BamHI, and then analyzed by 1% agarose gel electrophoresis. This revealed that EcoRI and Bg111 cleaved the plasmid at a single site to give a product having a size of 6.2 kb while BamHI did not cleave it.Since pUB110 used as the vector has a size of 4.5 kb and is cleaved by EcoRI, Bg111 and BamHI at a single site, the plasmid obtained from the large halo forming strain was found to be a recombinant DNA molecule having a donor chromosomai DNA fragment of about 1.7 kb size (i.e., the neutral protease gene of Bacillus amyloliquefaciens) inserted into the BamHI-cleaved site of pUB110. This recombinant plasmid was named pNP150.
The recombinant plasmid pNP150 thus obtained was used to transform Bacillus subtilis strain 1A20 (a stock strain maintained at the above Bacillus Genetic Stock Center, the Ohio State University) by the competent cell method. This was carried out according to the procedure of Anagnostopoulos and Spizizen [Anagnostopoulos, C., and Spizizen, J., J. Bacteriol., 81,741(1961)]. After about 5 yg of the recombinant DNA molelcule was incorporated, 50 il each of the culture medium (1 ml) was plated onto the TBAB agar media containing 0.8% casein and 40 g/ml kanamycin. All of the resulting kanamycin-resist ant transformants formed a large halo. Using this transformed strain, or Bacillus subtilis strain MT-0150 (Deposition No.FERM BP-425 dated 10th December 1983; a stock strain maintained in the Fermentation Research Institute of the Agency of Industrial Science and Technology, 1-1-3, Higashi 1-chome, Yatabe Machi, Tsukuba gun, Ibaraki-ken 305, Japan), plasmid pNP150 consisting of plasmid pUB110 and the neutral protease gene of Bacillus amyloliquefaciens joined thereto was prepared in the usual manner.
Example 2 (Production of human interferon-p in Bacillus subtilis according to the procedure of Figure 4 using an expression-secretion vector having an expression- and secretion-related DNA base sequence derived from the neutral protease gene) Plasmid pNP150 obtained in Example 1 was cleaved with the restriction endonuclease Pvul in the usual manner (Figure 4(a)) and the resulting linear plasmid was treated with exonuclease Ba131 to remove about 220 base pairs from each end of the linear plasmid. Thus, there was obtained an expression-secretion plasmid in accordance with the present invention [Figure 4(b)].
Separately, plasmid plF20 was cleaved with the restriction endonuclease Dpnl to obtain the gene coding for mature human interferon-p which was deprived of the promoter and secretion signal regions [Figure 4(c)]. Then, a recombinant DNA was prepared by joining this gene to the aforesaid plasmid pNP150 which was treated with Bal 31 [Figure 4(d)]. The aforesaid plasmid pit20 was created with synthetic DNA linkers in such a way that cleavage sites for the restriction endonuclease Sau3A and cleavage sites for the restriction endonuclease Dpnl were present at both ends of the DNA sequence coding for the mature protein of human interferon-p deprived of the signal portion involved in its secretion.
Thereafter, Bacillus subtilis strain 1A510 (a stock strain maintained in the above Bacillus Genetic Stock Center, the Ohio State University) used as the host was transformed with the aforesaid recombinant DNA, and cells having received the plasmid were selected on the basis of their kanamycin resistance. In screening kanamycin-resistant cells according to the colony hybridization technique, 32P-labeled DNA coding for human interferon-p was used as the probe.
The colonies giving a positive result in the colony hybridization test were cultured in a liquid medium.
Specifically, each colony was cultured at 30"C, with shaking, in the BY medium of which composition was 0.5% meat extract, 0.2% yeast extract, 0.2% NaCI, 1% polypeptone and 5 g/ml kanamycin. After about 14 hours, the culture medium was centrifuged at 10,000 g for 5 minutes and the supernantant was recovered. The human interferon-p activity of the supernatant was measured by using enzyme immunoassay.
The result of the assay revealed that Bacillus subtilis strain OZ361 (Deposition No. FERM BP-683 dated 20th December 1984; a stock strain maintained in the above Fermentation Research Institute of the Agency of Industrial Science and Technology) produced an interferon having the activity of 4.0 x 104 U/ ml into the culture medium. A plasmid was prepared from this strain and named pOZ361.
This plasmid pOZ631 had the restriction endonuclease cleavage sites shown in Figure 4. The base sequence of one strand of the junction between human interferon-p DNA and pNP150 is shown in Figure 5.
When this plasmid was used again to transform Bacillus subtilis strain 1A510, all of the resulting kanamycin-resistant colonies were found to be productive of human interferon-p. One of these colonies was cultured at 30"C for 18 hours in the aforesaid medium further containing 10 nM phenyl-methanesuifonyl fluoride. Thus, 105 U of human interferon-p was accumulated in each milliliter of the supernatant of the culture medium.
Example 3 (Preparation of the a-amylase gene devoid of its expresssion- and secretion-related DNA sequence according to the procedure of Figure 4) 10 jffi9 of plasmid pTUB4 containing the Bacillus subtilis amylase gene [Takeichi et al., Agric. Biol.
Chem., 47, 159-161(1983)] was digested at 37"C with 10 units of the restriction endonuclease Alul (Takara Shuzo Co.) and 10 units of the restriction endonuclease EcoRI (Takara Shuzo Co.) for 1 hour [Figure 6(a)].
In addition to the plasmid and the endonucleases, the reaction system contained 10mM MgCI2 and 150mM NaCI in a 10mM Tris-HCI buffer solution (pH 7.5).
As a result of the reaction, the fore part (about 400 base pairs) of the DNA fragment containing the CL- amylase gene devoid of its region involved in the expression of the gene and the secretion of the protein thus produced was obtained. This DNA fragment was separated by gel electrophoresis using 1.0% lowmelting agarose (BRL, Inc.) and then recovered from the gel.
The DNA fragment was purified by extraction with phenol and extraction with chloroform, and then recovered by precipitation with ethanol.
The recovered DNA fragment was dissolved in 10 il of a 50mM Tri-HCI buffer solution (pH 7.5) and used as a DNA fragment (hereinafter referred to as fragment A) consisting of the fore part of the a-amylase gene devoid of its region involved in the expression of the gene and the secretion of the protein thus produced.
Plasmid pUC13 (PL-Pharmacia Biochemical Co.) was completely cleaved with the restriction endonuclease Smal (Takara Shuzo Co.) and the restriction endonuclease EcoRI (Takara Shuzo Co.) [Figure 6(b)], and then treated with Escherichia coli alkaline phosphatase (Takara Shuzo Co.) to hydrolyze the terminal phosphate. Thereafter, the resulting vector DNA and fragment A were joined together by means of T4 ligase [Figure 6(c)].
The cleavage of plasmid pUC13 was carried out by treating 5 Ag of plasmid pUC13 at 37"C with 10 units of Smal and 10 units of EcoRI for 1 hour. In addition to the plasmid and the endonucleases, the reaction system contained 10mM MgCI2 and 150mM NaCI in a 10mM Tris-HCI buffer solution (pH 7.5).
The resulting Smal- and EcoRI-cleaved pUC13 DNA was successively purified by extraction with phenol, extraction with ether, and precipitation with ethanol.
The pUC13 DNA thus obtained was dissolved in 20 il of a 50mM Tris-HCl buffer solution (pH 7.5) and used as vector DNA.
This vector DNA and fragment A were joined together by means of the T4 ligase of Escherichia coli.
The reaction system contained 10 iil of fragment A (in solution in the aforesaid buffer solution), 10 pl of the vector DNA (in solution in the aforesaid buffer solution), 5 units of the T4 DNA ligase of Escherichia coli, 6.6mM MgCI2, 10mM dithiothreitol and 2mM ATP (adenosinetriphosphate) in a 66mM Tris-HCI buffer solution (pH 7.5). The reaction was carried out at 15"C for 2 hours.
Using the recombinant DNA obtained as a result of the reaction, Escherichia coli strain K-12 (JM101) (BRL, Inc.) used as the host was transformed in the usual manner [Mandei, M., and A. Higa, J. Moi. Biol., 53 154(1970)]. After transformation, ampicillin-resistant transformants were selected by use of the TBAB medium (Difco) containing ampiciliin at a final concentration of 50 Fg/ml.
One of the ampicillin-resistant strains thus obtained was cultured in 50 ml of the Penassay medium (Difco) at 37"C for 14 hours. Thereafter, the cells were collected and used in the preparation of a plasmid by the alkaline method [Birnboim, H.C., et al., Nucleic Acids Res., 7, 1513(1979)].
The nucleotide sequence of the junction between plamid pUC13 and fragment A is shown in Figure 2.
When the plasmid thus obtained was digested with the restriction endonucleases BamHI and EcoRI, about 400 and about 2700 base pair DNA fragments were noted. This demonstrates that the aforesaid plasmid is a recombinant plasmid having fragment A (comprising about 400 base pairs) inserted between the Smal and EcoRI cleavage sites of plasmid pUC13. This recombinant plasmid will hereinafter referred to as pAM11. Then, 50 Fg of plasmid pAM11 was cleaved with 10 units of the restriction endonuclease BamHI and 10 units of the restriction endonuclease EcoRI to obtain a DNA fragment comprising about 400 base pairs (hereinafter referred to as fragment B) [Figure 6(d)].
This fragment B contains the entire DNA sequence of the fore part of the a-amylase gene devoid of its region involved in the expression of the gene and the secretion of the protein thus produced.
Separately, 50 Fg of the aforesaid plamid containing the Bacillus subtilis a-amylase gene was cleaved with the restriction endonucleases EcoRI and Pvull to prepare a DNA fragment comprising about 1300 base pairs and containing the hind part of the a-amylase gene (hereinafter referred to as fragment C) [Figure 6(e)].
Furthermore, fragment B and fragment C were joined to plasmid pUC12 DNA (Pharmacia Co.) [Figure 6(g)), which had been cleaved with the restriction endonucleases BamHI and Hincll (Takara Shuzo Co.) and then treated with Escherichia coli alkaline phosphatase [Figure 6(f)].
The cleavage and phosphatase treatment of plasmid pUC12 [Figure 6(f)1 were carried out as follows: 5 iLg of plasmid pUC12 DNA was treated at 37"C with 10 units of Hincll and 10 units of BamHI for 1 hour, purified by extraction with phenol and then recovered by precipitation with ethanol. To the recovered DNA were added Escherichia coli alkaline phosphatase (5 units) and a Tris-HCI buffer solution (pH 8.0) in a final concentration of 0.1 M. The resulting reaction mixture was incubated at 65"C for 2 hours.
After completion of the reaction, DNA was purified by extraction with phenol and extraction with ether, and then recovered by precipitation with ethanol. Subsequently, the recovered DNA was redissolved in 20 l of a 50mM Tris-HCI buffer solution.
Using the same procedure as described above, 2 ,ug of fragment B and 2 pWg of fragment C, dissolved in Tris-HCI buffer solution (pH 7.5), were joined to 1 Fg of the pUC12 DNA so treated by means of the T4 ligase of Escherichia coli (Takara Shuzo Co.).
Using the recombinant DNA thus obtained, Escherichia coli strain K-12 (JM101) was transformed to obtain transformants having resistance to ampicillin. Then, plasmid DNA was prepared from one of these ampicillin-resistant strains by the alkaline method.
The resulting plasmid DNA was treated with the restriction endonuclease EcoRI alone or with the restriction endonucleases BamHI and Hindlll. Thus, it was confirmed that the treatment with EcoRI gave a DNA fragment of about 4500 base pairs cleaved at a single site, while the treatment with BamHI and Hindlll (Takara Shuzo Co.) gave a DNA fragment of about 1800 base pairs and a- DNA fragment of about 2700 base pairs.
Since pUC12 used as the vector comprises about 2700 base pairs, it was confirmed from a synthetic consideration of the above results that the resulting plamid (hereinafter referred to as pAM29) was a recombinant plasmid in which the a-amylase gene devoid of its region involved in the expression of the gene and the secretion of the protein thus produced was inserted between the BamHI and Hindlll cleavage sites of pUC12.
The junctions between the region containing the a-amylase gene and pUC12 in this plasmid pAM29 are shown in Figure 3. Thus, cleavage sites for the restriction endonucleases EcoRI, Smal and Sstl are present upstream of the amino end of the a-amylase gene portion of plasmid pAM29 DNA, and cleavage sites for Hindlll and Pvull are present downstream of the carboxyl end thereof.
Example 4 (Production of oi-amylase according to the procedure of Figure 6 using an expression-secretion vector of the present invention having an expression- and secretion-related DNA sequence originating from Bacillus am yloliquefaciens) 10 Wg of the plasmid pNP150 DNA obtained in Example 1 was treated at 37"C with 10 units of the restriction endonuclease Pvul (Boehringer Mannheim A.G.) for 1 hour. The resulting Pvul-cleaved pNP150 DNA was extracted three times with phenol, extracted with ether to remove any remaining phenol, and then recovered by precipitation with ethanol [Figure 6(h)].
Subsequently, the recovered DNA was treated at 30 C with 10 units of exonuclease Bal31 (Boehringer Mannheim A.G.) for 50 minutes [Figure 6(i)]. In addition to the DNA and the exonuclease, the reaction system contained 1mM EDTA, 12mM CaCI2, 12mM MgCI2 and 60mM NaCI in a 20mM Tris-HCI buffer solution (pH 8.1). After completion of the reaction, DNA was purified by extraction with phenol and extraction with ether, and then recovered by precipitation with ethanol. The recovered DNA (hereinafter referred to as the nuclease-treated DNA) was dissolved in 50 Al of a 50mM Tris-HCI buffer solution (pH 7.5).
It was confirmed by 1% agarose gel electro-phoresis that the nuclease-treated DNA was a mixture of DNA fragments cut into varying lengths by the action of the exonuclease.
Separately, 5 yg of pAM29 obtained in Example 3 was treated at 37"C with 10 units of the restriction endonuclease Sstl (BRL Inc.) and 10 units of the restriction endonuclease Pvull (Boehringer Mannheim A.G.) for 1 hour [Figure 6(j)]. Thus, a DNA fragment containing the amylase gene comprising about 2000 base pairs and devoid of its region involved in the expression of the gene and the secretion of the protein thus produced (hereinafter referred to as the structure gene for a-amylase) was obtained. This DNA fragment was separated, purified and recovered in the same manner as described above.Subsequently, both ends of this DNA fragment were treated with the T4 polymerase of Escherichia coli (Takara Shuzo Co.) to generate flush ends [Lehman, J.R., Method in Enzymology, 29, 46-53(1978)].
The resulting purified DNA fragment containing the structure gene for amylase and the previously prepared Ba131-treated pNP150 were joined together by means ofT4 ligase [Figure 6(k)].
As described above, the nuclease-treated DNA previously prepared from plasmid pNP150 was a mixture of DNA fragments varying in length, so that the resulting recombinant DNA was also a mixture of DNA molecules varying in length. Using the recombinant DNA mixture thus obtained, Bacillus subtilis was transformed according to the aforementioned protoplast method of Chang.
The protoplasts were regenerated in a medium containing 100 pg/ml kanamycin sulfate (Boehringer Mannheim A.G.) and soluble starch (Wako Pure Chemicals Co.) at a final concentration of 1%. As the host for transformation, Bacillus subtilis strain 1A289 lacking the ability to produce amylase (a stock strain maintained in the above Bacillus Genetic Stock Center, the Ohio State University) was used. Transformants producing and secreting amylase were selected according to the iodine/potassium iodide method in which the formation of a halo by the decomposition of starch was used as an index [J. Bacteriol., 179, 416-424(1974)]. Thus, several tens of transformed strains producing and secreting amylase were obtained.These transformed strains were shake cultured in the BY medium at 37C for 8 hours and the supernatant of each culture medium was assayed for (x-amylase activity. The activity was assayed by examining the formation of reducing groups from soluble starch with the aid of dinitrosalicylic acid (Biochemical informationll, Boehringer Mannheim, p. 28-30).
Then, plasmids were prepared from one of the transformed strains productive of amylase according to the aforementioned alkaline method. Each of the plasmids thus obtained was cleaved with the restriction endonucleases BamHI, Saml and Sstll, and then analyzed by 1% agarose gel electrophoresis. This revealed that BamHI cleaved the plasmids at a single site, Smal at two sites, and Sstll at two sites, respectively.
It is known that the DNA portion of pNP150 involved in the expression of the neutral protease gene and the secretion of the protein thus produced is cleaved by the restriction endonuclease Sstll at a single site and is not cleaved by BamHI or Smal. It is also known that the structure gene for a-amylase is cleaved by the restriction endonuclease Smal at a single site and is not cleaved by BamHI or Sstll. Moreover, it has been shown that the structure gene for a-amylase prepared from pAM29 by treatment with the restriction endonuclease Sstl and Hindlll and comprising about 2000 base pairs has cleavage sites for the restriction endonucleases Smal and BamHI immediately upstreak of its amino end.It was confirmed from these facts that the plasmids obtained from the transformed strain secreting a-amylase were recombinant DNAs containing the region necessary for the expression of the neutral protease gene and the secretion of the protein thus produced, and the DNA fragment obtained from pAM29. In these recombinant DNAs, the junction between (a) the DNA portion involved in the expression of the neutral protease gene and the secretion of the protein thus produced; and (b) the structure gene for amylase had the DNA sequence shown in Figure 1. This means that, if a DNA fragment coding for a desired protein is inserted into these recombinant DNAs at one of the restriction endonuclease cleavage sites of the DNA sequence shown in Figure 1, the ability to produce a-amylase is lost and transformants productive of the desired protein can be readily selected. One of these recombinant DNAs was named pNPA74 as the expression-secretion vector of the present invention.
Example 5 (Expression of the human interferon-gb gene and secretion of human interferon-p in Bacillus subtilis according to the procedure of Figure 7 using the expression-secretion vector pNPA74) First of all, plasmid plF20 containing the structure gene for human interferon-p and used in Example 2 was cleaved with the restriction enzyme Sau3A (Takara Shuzo Co.) to prepare a DNA sequence comprising about 500 base pairs and coding for the mature human interferon-p protein devoid of the signal portion involved in its secretion (hereinafter referred to as the structure gene for human interferon-p) [Figure 7(a)].
Then, the expression-secretion vector pNPA74 (5 yg) was completely cleaved with the restriction endonuclease BamHI [Figure 7(b)]. Using the T4 ligase of Escherichia coli, the structure gene for human interferon-p (5 pg) was inserted thereinto to form a recombinant DNA [Figure 7(c)]. Furthermore, Bacillus subtilis strain 1A289 (a stock strain maintained in the above Bacillus Genetic Stock Center, the Ohio State Unviersity), which is a mutant strain unproductive of amylase, was transformed with the resulting recombinant DNA.As described in Example 4, the Bacillus subtilis strain 1A289 transformed with the recombinant DNA formed by inserting the DNA fragment coding for the structure gene for human interferon-p into pNPA74 at the BamHI cleavage site shown in Figure 1 did not have the ability to produce amylase and could be readily distinguished from thestrain transformed with pNPA74.
Fourteen strains thought to have been transformed with the recombinant DNA were shake cultured in the Penassay medium (Difco) at 30"C for 14 hours, and the supernatant of the culture medium was assayed for human interferon-p activity. This assay was made by an enzyme-immunoassay method using an antiserum against human interferon-p [Eiji Ishikawa (ed.), Enzyme-immunoassay, p.67 (1981)]. As a result, the transformed strains were found to secrete human interferon-p. Its activity was 2.5 x 104 U/ml.
Thereafter, a plasmid was prepared from one human interferon-p producing strain (B. subtilis Nip174) according to the above-described alkaline method. When the resulting plasmid DNA was treated with the restriction endonuclease Pvull, it was cleaved at two sites.
It is known that the structure gene for human interferon-p is cleaved by the restriction endonuclease Pvuli at a single site. It is also known that pNPA74 DNA used as the expression-secretion vector is simi larly cleaved by the restriction endonuclease Pvull at a single site. It was confirmed from these facts that the plasmid obtained from the human interferon-p producing strain was a recombinant DNA having the structure gene for human interferon-p incorporated into the expression-secretion vector pNPA74 in accordance with the present invention. The plasmid thus obtained will hereinafter be referred to as pNA1 74.
Conventionally, human interferon-p has been produced and recovered, with much labor, from human tissues or cultured cells originating from human tissues. However, the recombinant DNA of the present invention makes it very easy to effect the production and secretion of human interferon-p in Bacillus subtilis.
As used herein, one unit of human interferon-p corresponds to 1 x 10-6 mg.
Example 6 (Formation of an expression-secretion vector of the present invention according to the procedure of Figure 8) Plasmid pNP150 obtained in Example 1 was cleaved with the restriction endonuclease Sphl to obtain a large fragment of about 5.4 kb size and a small fragment of about 1 kb size [Figure 8(a)].
This large fragment was separated and recovered by low-melting agarose gel electrophoresis. The recovered large DNA fragment was purified in the usual manner by column chromatography, extraction with phenol, extraction with ether, and precipitation with ethanol.
0.1 Fg of the purified large DNA fragment was treated with 5 units of T4 ligase to form a circular plasmid (Figure 8(b)). In addition to the large DDA fragment and the ligase, the ligation reaction system contained 6.6mM MgCl2, 10mM dithiothreitol and 2mM ATP in a 66mM Tris-HCI buffer solution (pH 7.5). The reaction was carried out at 150C for 3 hours.
Using this reaction mixture, Bacillus subtilis strain 1A510 (a stock strain maintained in the above Bacillus Genetic Stock Center, the Ohio State University) was transformed according to the protoplast method.
According to the alkaline method, a plasmid was extracted from the resulting kanamycin-resistant transformed strain MT-1150 (Deposition No. FERM BP-722 dated 27th February 1985; a stock strain maintained in the above Fermentation Research Institute of the Agency of Industrial Science and Technology).
Then, its physical map was constructed by means of various restriction endonucleases.
As a result, it was confirmed that plasmid pES150 thus obtained had the DNA sequence involved in the expression of the neutral protease gene of Bacillus amyloliquefaciens and the DNA sequence coding for its prepro-peptide (i.e., the DNA sequence set forth in claim 7), and was deprived of the DNA sequence coding for mature extracellular neutral protease, which is located downstream of the Sphl cleavage site present in the DNA sequence coding for the cleavage site of propeptide.
As shown in Figure 8, this plasmid has a single cleavage site for Sphl and constitutes an expressionsecretion vector with which a recombinant DNA useful for the production and secretion of a desired pro tein can be easily constructed by inserting a DNA sequence containing the region coding for the desired protein into the aforesaid cleavage site, either directly or indirectly with the aid of suitable linkers, so as not to cause any misreading of the codons.
Furthermore, 1 9 of pES150 was cleaved with Sphl to open the ring. The resulting linear pES150 fragment was treated with T4 polymerase having exonuclease activity to remove the single-stranded termini, and the DNA sequence shown in Figure 1 was joined thereto by flush-end ligation. Thus, there were constructed the expression-secretion vectors pSB150 and pBS150 shown in Figure 2 [Figure 8(cell In these expression-secretion vectors, the DNA sequence shown in Figure 1 is located downstream of the DNA sequence coding for the cleavage site of pro-peptide of neutral protease, and cleavage sites for the restriction endonucleases Smal and BamHI are present in the DNA sequence.
Accordingly, a recombinant DNA useful for the production and secretion of a desired protein can be easily constructed by inserting a DNA sequence containing the region coding for the desired protein into one of the cleavage sites, either directly or indirectly with the aid of suitable linkers, so as not to cause any misreading of the codons.
The formation of pSB150 and pBS150 from pES150 is more concretely described hereinbelow.
1 pLg of pES150 was cleaved with Sphl in the usual manner to open the ring. The resulting linear pES150 was purified by extraction with phenol, extraction with ether, and precipitation with ethanol.
After drying, the linear pES15O was dissolved in 32 Fl of distilled water. To the resulting solution were added 4 Fl of a 104old concentrated T4 polymerase buffer solution, 2 'LI of 2mM a-NTP, and 3 units ofT4 DNA polymerase (Takara Shuzo Co.). This reaction mixture was incubated at 37 C.
After 30 minutes, 150 'LI of a DNA buffer solution (containing 10mM KCI and 0.1mM EDTA in a 10mM Tris-HCI buffer solution, pH 8.0) was added thereto, and the DNA was purified by extraction with phenol, extraction with ether, and precipitation with ethanol. After drying, the DNA was dissolved in 50 ,al of a Tris-HCI buffer solution (50mM, pH 7.5) and 10 pl of the resulting solution was used in the following procedure.
Specifically, the DNA sequence shown in Figure 1 was synthesized separately. 0.2 9 of this DNA sequence was added to 0.1 'il of the above DNA solution and joined to the DNA by means of T4 ligase.
In addition to the DNA, the reaction system contained 20 units of T4 ligase (Takara Shuzo Co.), 6.6 mM MgCl2, 10mM dithiothreitol and 2mM ATP in a 66mM Tris-HCI buffer solution (pH 7.5). The reaction was carried out at 4"C for 20 hours. The reaction mixture was adjusted to a total volume of 50 FI.
Using 20 Fl of the reaction mixture thus obtained, Bacillus subtilis strain 1A510 was transformed according to the protoplast method. Plasmids were prepared from the resulting kanamycin-resistant strains and their physical maps were constructed in the usual manner. As a result, it was found that these plasmids had no cleavage site for Sphl and had single cleavage sites for Smal and BamHI.
Thus, there were two types of plasmids according to the direction in which the DNA sequence shown in Figure 1 was inserted thereinto. These plasmids were named pSB150 and pBS150, respectively.
Example 7 (Production and secretion of a-amylase according to the procedure of Figure 9 using the expressionsecretion vector pES150) 1 CLg of pAM29 obtained in Example 3 was cleaved with the restriction endonucleases Sstl and Hindlll in the usual manner. The resulting 1.8 kb fragment containing the structure gene for o!-amylase was separated and recovered by low-melting agarose gel electrophosresis, and then purified by extraction with phenol, extraction with ether, and precipitation with ethanol [Figure 9(a)].
After drying, the purified DNA fragment was dissolved in 32 pl of distilled water and treated with T4 DNA polymerase under the same conditions as in Example 6 to remove the single-stranded termini and thereby generate flush ends [Figure 9(b)]. After treatment with T4 DNA polymerase, the DNA was purified again in the same manner as in Example 6.
After drying, the purified DNA was dissolved in 20 'LI of a 50mM Tris-HCI buffer solution (pH 7.5) and 10 (zl of the resulting solution was used in the following procedure. Separately, pES150 (0.5 'lug) was cleaved with the restriction endonuclease Sphl to open the ring, and then treated with T4 polymerase in the same manner as in Example 6 to generate flush ends. This plasmid and the above purified DNA were joined together by means of T4 ligase [Figure 9(c)]. The reaction conditions were the same as those employed in Example 6.
Using the reaction mixture thus obtained, Bacillus subtilis strain 1A289 (a stock strain maintained in the above Bacillus Genetic Stock Center, the Ohib State University), which is a strain unproductive of a-amy- lase, was transformed according to the protoplast method. Thereafter, the protoplasts were plated onto the DM-3 regeneration medium containing 1% soluble starch and 150 Fg/ml kanamycin, and cultured at 37"C for 24 hours. Then, a potassium iodide solution was sprayed over the plate and 50 transformed strains forming a halo were obtained. A plasmid was prepared from one of the transformed strains, or strain MT-33, and its physical map was constructed. Thus, it was found that this plasmid (named pPA33) had an about 1.8 kb DNA sequence containing the structure gene for oi-amylase, the DNA sequence being joined to pES150 on the downstream side of the DNA sequence coding for the pro-peptide of neutral protease.
pPA33 also has the DNA sequence shown in Figure 1, which is located upstream of the structure gene for amylase and joined to the DNA sequence coding for the cleavage site of pro-peptide of neutral protease. Cleavage sites for the restriction endonucleases Smal and BamHI are present in this DNA sequence.
Using the BY medium [H. Uehara et al., J. Bacteriol., 119, 82(1974)], Bacillus subtilis strain MT-33 having this recombinant DNA pPA33 was cultured at 37"C for 18 hours. When the supernatant of the culture medium was assayed for a-amylase activity according to the starch-iodine method [H. Fuwa, J. Biochem.
(Tokyo), 41, 583(1954)], the enzyme was found to be secreted in an amount of 2500 units per milliliter of the supernatant. The activity of strain 1A289 used as a control was below the detection limit.
Example 8 (Production and secretion of human interferon-p) Plasmid plF20 used in Example 2 and containing the structure gene for human interferon-p was cleaved with the restriction endonuclease Sau3Al to obtain a 0.5 kb DNA sequence coding for the mature protein of human interferon-p (i.e., the structure gene for hlFN-p).
Using T4 ligase, 1 9 of the structure gene for hlFN-p was inserted into pPA33 (1 9) constructed in Example 7 at its BamHI cleavage site. The reaction conditions were the same as those employed in Example 6.
Using the reaction mixture thus obtained, Bacillus subtilis strain 1A510 was transformed according to the protoplast method. Then, the protoplasts were cultured on the DM-3/kanamycin (150 Fg/ml) medium containing 1% of starch and 20 colonies not forming a large halo were selected.
These transformed strains were cultured in the BY medium (containing 5 Fg/ml kanamycin) at 30"C for 18 hours, and the supernatant of each culture medium was assayed for hlFN-p activity by enzyme immunoassay. As a result, the transformed strain #25 was found to secrete 5 x 104 U/ml of hIFN-p into the supernatant of the culture medium.
When a plasmid was prepared from this strain and examined, it was confirmed that this plasmid was that shown in Figure 10 (named pPIF25).
Using the BY medium (containing 5 Fg/ml kanamycin) having 10mM PMSF added thereto, the transformed strain #25 was cultured at 30"C for 18 hours. As a result, the supernatant exhibited an hlFN-p activity of as high as 5 x 105 U/ml.

Claims (31)

1. An expression-secretion vector comprising (a) a vector portion containing a region involved in its replication in host microorganisms; (b) a DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced; and (c) a terminus and/or one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted into or joined to said vector on the downstream side of said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced.
2. An expression-secretion vector as claimed in claim 1 wherein said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced is a DNA sequence containing a region involved in the expression of a gene coding for the extracellular enzyme of a bacterium of the genus Bacillus and the secretion of the enzyme thus produced.
3. An expression-secretion vector as claimed in claim 2 wherein said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced is a DNA sequence containing a DNA sequence involved in the expression of the gene coding for the extracellular neutral protease of a bacterium of the- genus Bacillus and a DNA sequence coding for its prepro-peptide.
4. An expression-secretion vector as claimed in claim 3 wherein said bacterium of the genus Bacillus is Bacillus amyloliquefaciens.
5. An expression-secretion vector as claimed in claim 1 wherein said vector portion is derived from a member selected from the group consisting of plasmids, phages and derivatives thereof that are capable of replication in bacteria of the genus Bacillus.
6. An expression-secretion vector as claimed in claim 5 wherein said vector portion is derived from a member selected from the group consisting of plasmids pUB110, pTP5, pUC194, pE194, pSA0501, and pBD6, phages 15, per29, 105 and p11, and derivatives thereof.
7. An expression-secretion vector as claimed in claim 3 wherein said DNA sequence containing a DNA sequence involved in the expression of the gene coding for the extracellular neutral protease of a bacterium of the genus Bacillus and a DNA sequence coding for its prepro-peptide contains the whole or a part of the DNA sequence in which one of the strands comprises bases arranged in the following order:: 10 20 30 40 GGATCTTAAC AmTTCCCC TATCATTTTT CCCGTCTTCA 50 60 70 80 TTTGTCATT TTTCCAGAAA AAATCGTCAT TCGACTCATG 90 100 110 120 TCTAATCCAA CACGTCTCTC TCG G CTTATC CCCTGACACC 130 140 150 160 GCCCGCCGAC AGCCCGCATG GACGAATCTA TCAATTCAGC 170 180 190 200 CGCGGAGTCT AGOG TAT TGCAGAATGC GAGATTGCTG 210 220 230 240 GTTTATTATA ACAATATAAG TTTTCATTAT TTTCAAAAAG 250 260 270 280 GGGGATTTAT TGTGGGTTTA GGTAAGAAAT TGTCTAGTGC 290 300 310 320 TGTAGCCGCT TCCTTTATGA GTTTAACCAT CAGTCTGCCG 330 340 350 360 GGTGTTCAGG CCGCTGAGAA TCCTCAGCTT AAAGAAAACC 370 380 390 400 TGACGAATTT TGTACCGAAG CATTCTTTGG TGCAATCAGA 410 420 430 440 ATTGCCTTCT GTCAGTGACA AAGCTATCAA GCAATACTTG 450 460 470 480 AAACAAAACG GCAAAGTCCT TAAAGGCAAT CCTTCTGAAA 490 500 510 520 GATTGAAGCT GATTGACCAA ACGACCGATG ATCTCGGCTA 530 540 550 560 CAAGCACTTC CGTTATGTGC CTGTCGTAAA CGGTGTGCCT 570 580 590 600 GTGAAAGACT CTCAAGTCAT TATTCACGTC GATAAATCCA 610 620 630 640 ACAACGTCTA TGCGATTAAC GGTGAATTAA ACAACGATGT 650 660 670 680 TTCCGCCAAA ACGGCAAACA GCAAAAAATT ATCTGCAAAT 690 700 710 720 CAG G CG CTG G ATCATGCTTA TAAAGCGATC GGCAAATCAC 730 740 750 760 CTGAAGCCGT TTCTAACGGA ACCGTTGCAA ACAAAAACAA 770 780 790 800 AGCCGAGCTG AAAGCAGCAG CCACAAAAGA CGGCAAATAC 810 820 830 840 CGCCTCGCCT ATGATGTAAC CATCCGCTAC ATCGAACCGG 850 860 870 880 AACCTGCAAA CTGG GAAGTA ACCGTTGATG CGGAAACAGG 890 900 910 920 AAAAATCCTT GAAAAAGCAA AACAAAGTGG GCATGCCGCC 930 940 950 960 ACAACCGGAA CAGGTACGAC TCTTAAAGGA AAAACGGTCT 970 980 990 1000 CATTAAATAT TTCTTCTGAA AGCGGCAAAT ATGTGCTGCG 1010 1020 1030 1040 CGATCTTTCT AAACCTACCG GAACACAAAA TAATACGTAC 1050 1060 1070 1080 GATCTGCAAA ACCGCGAGTA TAACCTGCCG GGCACACTCG 1090 1100 1110 1120 TATCCAGCAC CACAAACCAG I | TTTACAACTT C where A, T, G and C represent adenine, thymine, guanine and cytosine, respectively.
8. An expression-secretion vector as claimed in claim 3 wherein a restriction endonuclease cleavage site present in the DNA sequence, which is contained in said DNA sequence coding for the prepro-peptide and which codes for the cleavage site of the propeptide or the neighborhood of the pro-peptide cleavage site, is used as one of said restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted into or joined to said vector.
9. An expression-secretion vector as claimed in claim 8 wherein said restriction endonuclease cleavage site present in said DNA sequence coding for the cleavage site of the pro-peptide is a cleavage site for the restriction endonuclease Sphl.
10. An expression-secretion vector as claimed in claim 1 wherein said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced is one obtained by (a) cleaving a DNA sequence containing a gene coding for an extracellular protein, which includes a DNA sequence containing a region involved in the expression of the gene and the secretion of the protein thus produced, at a restriction endonuclease cleavage site located downstream from the DNA sequence containing a region involved in the expression of the gene and the secretion of the protein thus produced; and then (b) digesting the end part of the cleaved DNA sequence, which is located downstream from the DNA sequence containing a region involved in the expression of the gene and the secretion of the protein thus produced, in such a way that the DNA sequence contain ing a region involved in the expression of the gene and the secretion of the protein thus produced is retained and the terminus so formed make it possible to join thereto a DNA sequence containing a gene coding for a desired protein.
11. An expression-secretion vector as claimed in claim 10 wherein said end part of the cleaved DNA sequence are digested with an exonuclease.
12. An expression-secretion vector as claimed in claim 3 wherein said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced is one formed by (a) cleaving a DNA sequence containing the extracellular neutral protease gene of a bacterium of the genus Bacillus at a restriction endonuclease cleavage site located downstream from a DNA sequence containing the region involved in the expression of the gene and the secretion of the extracellular neutral protease thus produced; (b) digesting the end part of the cleaved DNA sequence, which is located downstream from the DNA sequence containing the region involved in the expression of the gene and the secretion of the extracellular neutral protease thus produced, with an exonuclease in such a way that the DNA sequence containing the region involved in the expression of the gene and the extracellular neutral protease thus produced is retained; and then (c) joining to the downstream end of the digested DNA sequence a DNA sequence having a terminus-and/or one or more retriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto.
13. An expression-secretion vector as claimed in claim 12 wherein said DNA sequence having a terminus and/or one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto contains the following nucleotide sequence: 5' CGCCCGGGGATCCCC 3' 3' GCGGGCCCCTAGGGG 5'
14.An expression-secretion vector as claimed in claim 3 wherein said DNA sequence containing a region involved in the expression of a gene coding for a protein and the secretion of the protein thus produced is one formed by (a) cleaving a DNA sequence containing the extracellular neutral protease gene of a bacterium of the genus Bacillus at the cleavage site for the restriction endonuclease Sphl present in the DNA sequence which is contained in the extra-cellular neutral protease gene and which codes for the cleavage site of the pro-peptide; (b) treating the resulting single-stranded end located on the downstream side of the DNA sequence coding for the prepro-peptide of the neutral protease with an enzyme having exonuclease activity to generate a flush end; and then (c) joining to said flush end a DNA sequence having a terminus and/or one or more retriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto.
15. An expression-secretion vector as claimed in claim 14 wherein side DNA sequence having a terminus and/or one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto contains the following nucleotide sequence: 5' CGCCCGGGGATCCCC 3' 3' GCGGGCCCCTAGGGG 5'
16. An expression-secretion vector as claimed in claim 13 or 15 wherein the amylase gene of Bacillus subtilis deprived of the DNA sequence coding for its secretion signal region is joined to said vector through the medium of said DNA sequence having a terminus and/or one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto.
17. A recombinant DNA comprising an expression-secretion vector as claimed in claim 1 and a DNA sequence containing a gene coding for a desired protein which is inserted into or joined to said expression-secretion vector at said terminus or one of said restriction endonuclease cleavage sites.
18. A recombinant DNA as claimed in claim 17 which is constructed by (a) providing a plasmid comprising an extracellular enzyme gene of a bacterium of the gene Bacillus, which includes a DNA sequence containing a region involved in the expression of the extracellular enzyme gene and the secretion of the enzyme protein thus produced, and a vector portion containing a region involved in its replication in host microorganisms; (b) cleaving said plasmid at one of the restriction endonuclease cleavage sites located downstream from the DNA sequence containing a region involved in the expression of the extra-cellular enzyme gene and the secretion of the extra-cellular enzyme protein thus produced; (c) digesting the end parts of the cleaved plasmid containing separately the extracellular enzyme gene in such a way that both the DNA sequence containing a region involved in the expression of the gene and the secretion of the protein thus produced and the vector portion involved in its replication are retained; and then (d) inserting a DNA sequence containing a gene coding for a desired protein between the resulting ends of the digested plasmid.
19. A recombinant DNA as claimed in claim 17 which is constructed by (a) providing a plasmid comprising a extracellular neutral protease gene of a bacterium of the genus Bacillus and a vector portion containing a region involved in its replication in host microorganisms; (b) cleaving said plasmid at one of the restriction endonuclease cleavage sites located downstream from the DNA sequence containing the region involved in the expression of the extra-cellular neutral protease gene and the secretion of the extracellular neutral protease thus produced; (c) digesting the end parts of the cleaved plasmid containing separately the extracellular neutral protease gene with an exonuclease in such a way that the DNA base sequence containing a region involved in the expression of the gene and the secretion of the protein thus produced is retained; (d) inserting a DNA sequence, which has a one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto or joined thereto, between the resulting ends of the digested plasmid; and then (d) inserting a DNA sequence containing a gene coding for a desired protein into the inserted DNA base sequence at the one of the restriction endonuclease cleavage sites.
20. A recombinant DNA as claimed in claim 19 wherein said DNA sequence having one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto contains the following nucleotide sequence: 5' CGCCCGGGGATCCCC 3' 3' GCGGGCCCCTAGGGG 5'
21.A recombinant DNA as claimed in claim 17 which is constructed by providing a plasmid comprising a extracellular neutral protease gene of a bacterium of the genus Bacillus and a vector portion containing a region involved in its replication in host micro-organisms and having the cleavage sites for the restriction endonuclease Sphl present in the DNA sequence, which is contained in the extracellular neutral protease gene and which codes for the cleavage site of the pro-peptide of the neutral protease and downstream from the structure gene for the mature neutral protease; (b) deleting the DNA sequence between the Sphl cleavage sites, which contains the structure gene for the mature neutral protease, from the prasmid by cleaving the plasmid at the Sphl cleavage sites; (c) treating the resulting single-stranded ends of the cleaved plasmid with an enzyme having exonuclease activity to generate flush ends; (d) inserting a DNA sequence, which has one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto, between the resulting flush ends; and then (e) inserting into the inserted DNA sequence a DNA sequence containing a gene coding for a desired protein at the one of the restriction endonuclease cleavage sites.
22. A recombinant DNA as claimed in claim 21 wherein said DNA sequence having one or more restriction endonuclease cleavage sites at which a DNA sequence containing a gene coding for a desired protein can be inserted thereinto contains the following nucleotide sequence: 5' CGCCCGGGGATCCCC 3' 3' GCGGGCCCCTAGGGG 5'
23. A recombinant DNA as claimed in claim 21 wherein said gene coding for a desired protein is a gene coding for a extracellular a-amylase of a bacterium of the genus Bacillus deprived of the DNA sequence coding for its secretion signal region.
24. A recombinant DNA as claimed in claim 22 wherein the extracellular a-amylase gene of a bacterium of the genus Bacillus deprived of the DNA sequence coding for its secretion signal region is joined to said recombinant DNA downstream of said DNA sequence containing the base sequence shown in claim 22, and the human interferon-ss gene deprived of the DNA sequence coding for its secretion signal region is inserted into said DNA sequence at one of said restriction endonuclease cleavage sites.
25. A method of producing proteins which comprises transforming a bacterium of the genus Bacillus with a recombinant DNA, culturing the resulting transformed strain, and then recovering the desired protein from the culture medium.
26. A method of producing proteins as claimed in claim 25 wherein a recombinant DNA as claimed in claim 17 is used as said recombinant DNA.
27. A method of producing proteins as claimed in claim 25 wherein a recombinant DNA as claimed in claim 23 is used as said recombinant DNA.
28. A method of producing proteins as claimed in claim 25 wherein a recombinant DNA as claimed in claim 24 is used as said recombinant DNA.
29. An expression-secretion vector substantially as hereinbefore described.
30. A recombinant DNA substantially as hereinbefore described.
31. A method of producing proteins substantially as hereinbefore described.
GB8601033A 1985-01-18 1986-01-16 Expression-secretion vector for gene expression and protein secretion, recombinant dna including the vector, and method of producing proteins by use of the re Expired GB2171703B (en)

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EP0351717A2 (en) * 1988-07-21 1990-01-24 Henkel Kommanditgesellschaft auf Aktien Plasmid for production of alpha-amylase in Bacillus
EP0402159A1 (en) * 1989-06-09 1990-12-12 MITSUI TOATSU CHEMICALS, Inc. Polypeptide having thrombin inhibiting activity and process for the manufacture thereof
EP0409098A1 (en) * 1989-07-19 1991-01-23 ENIRICERCHE S.p.A. The expression and secretion of mature human beta interleukin-1 in bacillus subtilis and means and methods for its achievement
WO1991002792A1 (en) * 1989-08-25 1991-03-07 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
US5084383A (en) * 1986-10-02 1992-01-28 Agency Of Industrial Science And Technology Bacillus subtilis strain whose extracellular protease activities are reduced, method for obtaining the strain and method for secreting proteins by using the strain
EP1195437A1 (en) * 2000-10-04 2002-04-10 Mitsui Chemicals, Inc. Process for the recombinant production of proteins using a Plac/Np hybrid promoter
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US5084383A (en) * 1986-10-02 1992-01-28 Agency Of Industrial Science And Technology Bacillus subtilis strain whose extracellular protease activities are reduced, method for obtaining the strain and method for secreting proteins by using the strain
EP0351717A3 (en) * 1988-07-21 1990-07-25 Henkel Kommanditgesellschaft auf Aktien Plasmid for production of alpha-amylase in bacillus
EP0351717A2 (en) * 1988-07-21 1990-01-24 Henkel Kommanditgesellschaft auf Aktien Plasmid for production of alpha-amylase in Bacillus
EP0402159A1 (en) * 1989-06-09 1990-12-12 MITSUI TOATSU CHEMICALS, Inc. Polypeptide having thrombin inhibiting activity and process for the manufacture thereof
US5166318A (en) * 1989-06-09 1992-11-24 Mitsui Toatsu Chemicals, Incorporated Polypeptide having thrombin inhibiting activity
EP0409098A1 (en) * 1989-07-19 1991-01-23 ENIRICERCHE S.p.A. The expression and secretion of mature human beta interleukin-1 in bacillus subtilis and means and methods for its achievement
US5437988A (en) * 1989-07-19 1995-08-01 Eniricerche S.P.A. Expression and secretion of mature human beta interleukin-1 in Bacillus subtilis and means and methods for its achievement
US5728571A (en) * 1989-07-19 1998-03-17 Eniricerche S.P.A. Expression and secretion of mature human beta interleukin-1 in Bacillus subtilis and means and methods for its achievement
WO1991002792A1 (en) * 1989-08-25 1991-03-07 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
US5352604A (en) * 1989-08-25 1994-10-04 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
EP1195437A1 (en) * 2000-10-04 2002-04-10 Mitsui Chemicals, Inc. Process for the recombinant production of proteins using a Plac/Np hybrid promoter
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