JPH0146111B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to molecular biology and in particular to so-called recombinant DNA. This invention relates to a method for producing a genetically modified bacterium in which a cloned foreign gene is introduced into a Gram-positive bacterium such as Bacillus subtilis to produce the gene product under controlled conditions. Collection of items will be promoted. This invention discloses a novel genetically engineered plasmid. Examples of these microorganisms have been deposited under the Treaty of Budapest at the American Type Culture Center (ATCC), Rockville, Maryland 20852. Plasmid pOG1196 is ATCC No. 31776; Plasmid pOG2165 is ATCC No. 31777; Plasmid
pOG2110 is ATCC No. 31778. As is well known, the amino acid sequence of a particular protein is determined by the code carried in the gene for that protein. Methsenjar RNA from DNA
During the process of translation to form proteins through It is placed at the corresponding position of the above protein chain. With the advent of recombinant DNA technology, genetic changes can be made artificially by introducing predetermined nucleotide sequences synthesized or isolated from one strain or species into the genes of another strain or species. I am now able to do it properly. A known nucleotide sequence can be selected such that a strain or species into which the nucleotide sequence is introduced will produce the protein encoded by the known nucleotide sequence as part of the process of translation. A strain or species that has undergone such manipulation also replicates the introduced nucleotide sequence in the normal course of replication. Recombinant DNA technology consists of isolating the appropriate DNA strand (cloning vector) and cutting the two strands of DNA of the cloning vector at the location where you want to insert the foreign DNA. To do this,
Certain types of proteins, so-called restriction enzymes, are typically used. Restriction enzymes will cut DNA at specific nucleotide sequences, but for some restriction enzymes, the cuts will not necessarily occur at the same position of the two intertwined DNA strands. In such cases, if two different types of DNA are cut in the same way, each cleaved end will be complementary to each other and, under appropriate conditions, will interlock with a parallel complementary end. These ends are then joined together by an enzyme (ligase). This means that the two
It allows even DNA fragments to be incorporated into a single DNA molecule. Once the DNA vector has been isolated and the foreign fragment inserted, the recombinant DNA is placed into a suitable host microorganism. In order for the host microorganism to replicate the inserted DNA, it is necessary to insert the recombinant DNA into the host microorganism so that it becomes part of the host microorganism's genetic system. Once the foreign DNA has been successfully integrated into the host microorganism and the host microorganism produces the product of the cloned gene, the desired product must be recovered. Prior to the present invention, recovering this product required killing the cells producing the desired product. Also, cells contain many different proteins, making methods of isolating the desired product difficult or complicated. Ultimately, the desired product is harmful to the host cell, especially if it is produced at high levels. In some cases this causes the cell to disintegrate, and in other cases the cell's defense mechanisms are activated to degrade the desired product. Most of the recombinant DNA research to date has been carried out in E. coli, which lives in the periplasmic space.
It is a type of Gram-negative bacterium that has a two-layered cell membrane that encloses the cells. Many of the products produced in E. coli are secreted into this periplasmic space. Only a small amount of product is secreted from living cells into the growth medium. On the other hand, for example, it is a member of the Gram-positive bacteria group that has only a single cell membrane. Bacillus subtilis
produces large amounts of protein, which is secreted directly into the growth medium. A common research method for gene cloning in E. coli is Bacillus subtilis (B.
However, attempts to induce B. subtilis to produce useful products from foreign genes and secrete them into the growth medium have not been successful. Bacillus subtilis is somewhat preferred over E. coli because of its high efficiency of plasmid-mediated transformation and lack of pathogenicity. BRIEF DESCRIPTION OF THE DRAWINGS The objects of the invention will become apparent from the following description, taken in conjunction with the accompanying drawings. Figure 1 is an overall diagram showing a partial structural map of a DNA fragment containing the gene for β-lactamase obtained from Bacillus licheniformis; Figure 2 shows many of the fragments in Figure 1. Fig. 3 is an overall diagram showing the correspondence between the nucleotide sequence of the fragment and the amino acid sequence present in the protein encoded by the fragment; Fig. 3 is a typical appearance of a plate on which a Bacillus subtilis strain containing a recombinant plasmid is grown; FIG. 2 is a schematic diagram showing the plate β
- Shows the situation after performing the PVA assay to test lactamase activity. Figure 4 shows E. coli plasmid pOG2110.
Figure 5 is a general diagram illustrating the structure of B. subtilis plasmid pOG1196 and bifunctional plasmid pOG2165; Figure 5 shows a typical appearance of a plate on which various bacterial strains carrying recombinant plasmids are grown; Figure 6 shows the plate after PVA assay for testing β-clatamase activity; Figure 6 shows Bacillus licheniformis (B.
This is a diagram of the nucleotides that make up part of the Pen P gene. In one embodiment of the present invention, B. subtilis
A predetermined protein that is not native to B. subtilis is produced by expression in Bacillus subtilis.
B. subtilis with plasmid introduced
A suitable growth medium and conditions are provided for growing this strain. The plasmid is capable of replicating in the strain and contains the gene for the predetermined protein. The gene is located and oriented in the plasmid so that it is under the control of an operator, a promoter, and a ribosome binding site sequence. This protein is also placed under the control of transport machinery secreted by the host strain. Upon secretion, this protein is recovered from the growth medium. The method of the invention requires a cloning vector microorganism having a nucleotide sequence capable of initiating the transcription and translation process. The nucleotides providing the operator, promoter, and ribosomal binding site sequences may be present naturally in the vector, inserted as independent DNA fragments using recombinant DNA techniques, or inserted as part of the foreign DNA carrying the important gene. Department.
Foreign DNA, which will contain at least the structural gene for the desired protein product, is incorporated into a cloning vector so that it can be transcribed and translated under the control of an operator, a promoter, and a ribosome binding site sequence. For the inserted foreign DNA to be translated correctly, the nucleotides in the inserted DNA must be in the correct reading frame. Additionally, the cloning vector contains a sufficient number of nucleotides native to the host cell to ensure that the operator, promoter, and ribosomal binding site sequences are read and translated into the inserted foreign DNA in the correct reading frame. It is desirable or necessary to contain. According to the invention, the cloning vector utilized consists of a nucleotide sequence consisting of codons for a functional transport signal peptide sequence. Transport signal peptide sequences are usually short leader sequences of amino acids on newly created proteins. Although the mechanism by which transport signal peptide sequences operate is not yet fully understood, it is thought that transport proteins are excreted by cells, and as proteins are produced, they latch onto and pull the produced proteins out of the cytoplasm. ing. Once the transport function is performed by the transport signal peptide sequence, the transport sequence is removed by natural processes. According to the invention, foreign DNA can be inserted into a cloning vector such that the protein encoded by this DNA is transported according to the transport signal peptide sequence encoded by the signal codon. The thus cloned gene product is conveniently transported to a desired location and recovered therefrom. This has several advantages. Since the location is outside the cell, the host cell does not need to be destroyed for recovery of the gene product, so the gene product can be produced continuously without interruption. Also, since cells contain so many proteins, the ability to excrete the products of cloned genes greatly simplifies the isolation and purification of the products. After all, the product of a cloned gene can be harmful to the host cell, especially when produced at high levels, so the ability to retrieve the product of a cloned gene from outside the cell membrane often means that it does not harm the cell. Significantly, if there is no salvage capability, cells will produce defensive enzymes to degrade the gene product. The exact location at which the foreign DNA should be inserted into the cloning vector depends on the functioning of the transport signal peptide sequence. In some cases, the transport signal peptide sequence will immediately precede the foreign DNA, as part of the cell's gene itself, or will already be present on a plasmid. The signal sequence itself consists of a nucleotide sequence necessary for reading and translating foreign DNA. On the other hand, there are cases in which the transport signal peptide sequence should not be located immediately in front of the foreign DNA. In such cases it is necessary to generate the desired peptide sequence with a few more amino acids at the start site (encoded by the foreign codon) to provide the necessary reading function. The following examples are intended to illustrate other specific examples in which the invention may be used and are not intended to limit the scope of the invention. Example 1 Formation of a plasmid for producing an introduced predetermined protein that is not native to the host microorganism Providing a plasmid for producing a predetermined protein that is not native to the host microorganism For this purpose, we created a plasmid vector containing the B. licheniformis β-lactamase gene and used it to infect Bacillus subtilis (B.
subtilis) and E. coli. The above plasmid is 3.5Kb (Kirobase) containing the β-lactamase gene.
The EcoR-Sst fragment was purified and transferred to Escherichia coli (E.
coli) plasmid pOP1Δ6 (Gelfand et al., proc.
the Nat.Acad.Sci.USA (1978) 75 , 5869−5873)
2.1 Kb EcoR -Sst fragment with replication function. Appropriate E. coli CS412 cells (C600rk ^- mk ⁺ Pr-induced cells) were transformed with this, and after an appropriate growth time (90 minutes) for gene expression, Ap-resistant transformants were obtained. Obtained. Plasmids from three clones were obtained. The plasmid identified as pOG2110 was further characterized. Figure 4 is
Figure 2 details the locations of expected and observed restriction sites in pOG2110. To replicate pOG2110 in B. subtilis, the bifunctional replicon combines pOG2110 and the B. subtilis plasmid.
Formed using pOG1196. The formation of pOG1196 is summarized in Figure 4. Plasmids pC194 ( ^CmR ) and pUB110 ( ^KmR )
Chimeric plasmid (pCS832) containing the entire sequence of
first converted the two Mbo fragments of pC194 into BamH
generated by ligation with truncated pUB110. The resulting plasmid contains the Cm gene from pC194 and the Km (Nm) gene from pUB110. The size of this plasmid is 7.5Kb. Natural deletion mutant (plasmid pCS1006)
was obtained from one of the subclones. It has lost the Hpa site that occurred in pC194, and the pC194 replication region (Chang and Cohen, Molec.
Gen. Genet., (1979) 168 , 111-115). This is still pUB110
It retains a replication function and two resistance markers. Plasmid by recycling the largest Hpa fragment (3.6Kb) of pCS1006
pOG1196 was obtained. This plasmid provides only Cm-resistance and has the replication function from plasmid pUB110. A map of pOG1196 is shown in Figure 4. E. coli plasmid pOG2110 and Bacillus subtilis plasmid pOG1196 had two and three Pvu sites, respectively. Equal amounts of Pvu -cut pOG2110 and pOG1196 plasmid DNA were ligated and used to transform E. coli strain CS412. Cm-resistant clones were selected. All selected clones were also Ap-resistant. The composite plasmid pOG2165, isolated from one of the Cm-resistant Ap-resistant transformants, was further studied. this
A map of the 7.5Kb plasmid is shown in Figure 4. Plasmid pOG2165 replicates in both E. coli and Bacillus subtilis, and Cm-
and Ap − confers resistance. Bacillus subtilis and E. coli carrying plasmid pOG2165 are Bacillus licheniformis (B.
licheniformis) is resistant to ampicillin as a result of the production of the β-lactamase enzyme. This can be demonstrated by the PVA plate assay developed by Sherratt and Colling (see reference 9).
A positive result from such an assay is shown in FIG. When pOG2165 is grown in B. subtilis BD224,
A membrane bound and secreted exogenous β-lactamase is synthesized. The amount produced by this strain varies depending on the growth conditions used.
pOG2165 is also a Bacillus subtilis strain QB127 (Kunst et al.
Bio. Chemie. (1974) 56 1481-1490). QB127 is a sacU ^h mutated strain that overproduces several exoenzymes such as levansucrase, alpha-amylase, and extracellular proteolytic enzymes.
β- detected in cultures of QB127 (pOG2165)
Lactamase levels are BD224 under the same conditions
(pOG2165). The bifunctional plasmid pOG2165 itself contains the restriction enzyme Sst
, Hind , st and Bgl . Inserting DNA into the Bgl and Pst sites produces Bacillus licheniformis (B.
licheniformis) inactivates the β-lactamase gene and provides an easily confirmed phenotype to identify clones carrying the insert. If the exact open reading frame of the DNA sequence to be inserted is known, the first part of the β-lactamase exoenzyme can be prepared by cloning into the Bgl site.
Fusion proteins with 71 amino acid residues and a leader sequence can be created. The fusion protein produced by this method is secreted from Bacillus licheniformis cells due to the presence of a leader sequence at the amino terminus. In these respects, pOG2165 is a useful vector for cloning and efficient expression of foreign genes and subsequent secretion of the gene products in Bacillus subtilis and E. coli. On the other hand, if the exact reading frame of the DNA sequence to be inserted is known and the insertion is made at the Pst site, the fusion protein is made to accumulate in the host microorganism. Since the Pst confirmation site is located at the beginning of the nucleotide sequence encoding the signal peptide, only a portion of the nucleotide sequence is transcribed before encountering the foreign gene sequence. Even if the fusion protein is expressed, this portion of the signal peptide is insufficient to provide normal signal peptide secretion function. As a result, the product of the gene inserted into the Pst site accumulates in the host microorganism. Bacillus licheniformis
A portion of the nucleotide sequence consisting of the Pen P gene (B. licheniformis) is diagrammatically shown in FIG.
The β-lactamase promoter region is located between nucleotides 1 and 221, but the exact location is unknown. The nucleotides encoding the amino acids that make up the signal peptide begin at nucleotide 222 and end at nucleotide 323. As a result, the signal peptide consists of 34 amino acids. Pst confirmation site is nucleotide 25
9 and 264, ie, amino acids 13 to 15 of the signal peptide chain. Insertion of foreign DNA into this Pst site will form a fusion protein consisting of the foreign DNA product and the first 14 amino acids of the signal peptide chain. Although such a fusion protein is expressed, it will not be transported across the cell membrane. Successful secretion requires fusion with the entire signal peptide or at least the first 26 amino acid residues of the signal peptide chain. Example 2 Bacillus licheniformis (B.
licheniformis) is a secreted type (exoenzyme)
It produces large amounts of β-lactamase. Secretion of this protein is believed to be the result of interactions between the cell membrane and an amino acid leader sequence that facilitates transport of the protein across the monolayer cell membrane. Cloning the β-lactamase gene,
inserted into a plasmid that can replicate in Bacillus subtilis.
These plasmids were then transformed into B. subtilis
The cells were transformed into a host and secreted β-lactamase. The secretion consists of first expressing a gene foreign to Bacillus subtilis and transporting the gene product from the Bacillus subtilis cells to the medium or growth medium. Bacillus licheniformis (B.
To clone the β-lactamase gene from a B. licheniformis strain, total chromosomal DNA was isolated from B. licheniformis strain 749/C and cut with EcoR restriction endonuclease. Marmur's method (J.of
B. licheniformis by Molec.Biol. (1961) 3 , 208-18)
Chromosomal DNA was prepared from 749/C. Escherichia coli (E.
coli) plasmid pSC101 with Kupersztoch and
Helinski (Biochem.Biophys.Res.Commun.
(1973) 54, 1451-59). 3μg chromosomal DNA and 2μg
pSC101DNA was cut with the endonuclease EcoR and purified by Hershfield, et al.proc.Natl.Acad.Sci.
USA (1974) 71, 3455-59 by ligating with T4 DNA ligase and
Cohen, et al ^{. Proc} . Natl. Acad ^. Sci.;
USA (1972) 69, 2110-14. Transformants resistant to 10 μg/ml ampicillin were selected and one of the above transformants carrying recombinant plasmid pTB2 was further studied. Plasmid pTB2 carries a 4.2 Kb (kilobase pair) EcoR fragment on the pSC101 vector. This plasmid confers tetracycline (marker on pSC101) and ampicillin resistance to the host, and β-
It has been shown to produce lactamase gene products. The β-lactamase gene is located on the 4.2 kilobase pair EcoR fragment. Analysis of this fragment using various restriction enzymes and gene cloning leads to the structure of the gene for β-lactamase as partially mapped in FIG. The main sequence of β-lactamase obtained from B. licheniformis strain 749/C has been determined (RJ Meadway, Ph.D., University of Edinburgh dissertation). From the known amino acid sequence, Gly-Pro (116
−117 position) sequence is the nucleotide sequence GGN −
Corresponding to CCN, this is the endonuclease Sau 96
This is the confirmation sequence for (GGNCC). Similarly, the Trp-Pro (positions 222-223) sequence is encoded by the nucleotide codon TGG-CCN, with a central tetranucleotide sequence GGCC
was recognized, endonuclease Hae
(Roberts, DNA Insertion Elements, Plasm
ds, and Episomes, 1977, Bukhari, Shapiro.
and Adhya, Cold Spring Harbor Lad, p757)
cleaved by Supplier (New England Biolads, Inc., Beverly, Mass. 01915)
The 4.2 Kb cloned fragment was analyzed with a number of endonucleases as shown in FIG. 2 using conditions specified by (1978 catalog). The cut DNA was prepared by Sharp, et al.
Biochemistry (1973) 12, 3055-63 and analyzed on agarose gels and acrylamide gels (Maxam and Gillert, Natl.
Acad.Sci., USA (1973) 73 , 3942-46). The data for mapping are summarized in Figure 2. Sau 96 and Hae sites contain the complete β-lactamase gene sequence
It was localized to a 2.3Kb Pvu fragment. These two sites are separated by 320 nucleotides, which is consistent with the separation of 106 amino acids in the protein sequence data above. After fixing the nucleotide sequence containing the β-lactamase gene, the gene was transferred to various Bacillus plasmids and Bacillus subtilis.
subtilis)-E. coli fusion plasmid was used to introduce it into Bacillus subtilis. The plasmid is pUB110
and pC221 derived plasmid. The Bacillus subtilis strain containing this recombinant plasmid is not only resistant to ampicillin but also positive in the β-lactamase reaction on PVA plates. (See Figure 3) Furthermore, β-lactamase activity was detected in the culture even after the bacterial cells were removed. This activity indicates that not only was the foreign gene of Bacillus subtilis successfully expressed, but also that the protein was successfully transported through the cell membrane into the culture or growth medium. The Eco R fragment containing the β-lactamase gene was inserted into the Bacillus subtilis plasmid vector pUB110 using the method described above for E. coli.
(Gryczan and Dubnau, Proc. Natl. Acad. Sci.,
USA, (1978) 75 , 1428−1432) and pC221
(Ehrlich, Proc. Natl. Acad. Sci., USA (1977)
74, 1680-82) into each EcoR site.
Similarly, the 2.3Kb containing the β-lactamase gene
The Pva fragment was cloned into pUB 110 at the Pva and Tac sites. ligated DNA preparation
Chang and Cohen,
B. subtilis strains by the method of Molec. Gen. Genet., (1979) 168, 111-115 (see reference 7).
BD224 (recE4, trpC2, thr5) was transformed.
Transformants resistant to ampicillin were selected and tested on passage plates. β-lactamase production is detected by two methods. One is Sherratt
and Collins (J.Gen.Microbiol. (1973) 76 , 217
-230) and the other is the susceptibility plate assay developed by Ross and O Callaghan (Meth.in
Enzymology, (1975) 43 6.9-85). Bacillus subtilis clones resistant to ampicillin gave positive results to the above two methods. Furthermore, nucleotide-lactamase activity was also detected in the medium after bacterial cells were removed. This indicates that β-lactamase was not only produced but also excreted from B. subtilis. Keggins Proc.Natl.Acad.Sci.USA describes the method of cloning a foreign gene into Bacillus subtilis and allowing the gene to express functionally as an intracellular protein.
(1978) 75 , 1423-1427.
However, in this study, the cloned genes were usually derived from wild type B. subtilis.
It has not been demonstrated that Bacillus subtilis can successfully express genes that code for enzymes present in B. subtilis cells and secrete the products of these genes. do not have. The study of the β-lactamase gene demonstrated by this example shows for the first time that a new function, the β-lactamase production function, can be introduced into Bacillus subtilis by gene cloning techniques. Furthermore, this example is the first to demonstrate that a foreign gene product can be secreted as a foreign protein (exoprotein) through the barrier of the Bacillus subtilis cell membrane. Beta-lactamase, a commercially available and useful product, is produced by strains that cannot otherwise produce this enzyme. Example 3 The B. licheniformis β-lactamase gene described in Example 2 was also inserted into a plasmid capable of replication in E. coli. These plasmids were transformed into an E. coli host using the same method described for B. subtilis in Example 2. E. coli cells containing these plasmids are B. licheniformis β-
Resistant to ampicillin as a result of lactamase enzyme production. This is Sherratt and
as demonstrated by the PVA plate assay developed by Collins (see reference 9). When a plasmid carrying the Bacillus licheniformis β-lactamase gene is grown in E. coli, the secreted form of the β-lactamase is not transported into the medium as in Bacillus subtilis. Since E. coli has a cell wall surrounding the bacterial cell membrane, foreign protein products are transported into the periplasmic space that separates the cell membrane and cell wall. β-
The lactamase exoenzyme accumulates in the interstitial space around the protoplasm and is then recovered by a suitable method. Example 4 Bacillus licheniformis (B.
β-lactamases of B. licheniformis are not the only source of signal peptide sequences that can function in B. subtilis or E. coli. As mentioned above, many proteins (particularly in the mature nucleus) are transported across the cell membrane. The exact amino acid sequence of the “signal region” actually differs depending on the various proteins transported, but the folded structure of these signal regions (Chou and
Fasman Ann.Rev.Biochem.(1978) 47 , 251~
276 laws) are very similar. Although the requirement for a transport signal peptide sequence can thus be met by non-Bacillus signal peptides, the DNA fragment encoding the signal sequence must be correctly positioned downstream from the Bacillus promoter and ribosome binding site sequence. positioned. One such mature nuclear export signal sequence that can be used for the desired gene product is the signal or presequence that precedes the insulin B chain. (The various insulin chains A, B, and C are made as a single polypeptide, assembled, processed in the endoplasmic reticulum, and excreted by the cell.) However, the insulin precursor sequences contain foreign DNA. There are no convenient restriction sites immediately following the signal peptide sequence that can be used to join the cloned gene.
However, the last five nucleotides of the insulin signal sequence are AGGCT and the first nucleotide of the insulin B chain itself is a T. These six nucleotides are actually AGGCTT, which differs by one nucleotide from the confirmed sequence AAGCTT because of the restriction enzyme Hind. This enzyme cleaves between two As.
Foreign DNA uses the same enzyme Hind or
Hind site bifunctional linker (half Hind
When cloned or isolated using a site bifunctional linlcer), the foreign
When DNA is inserted, the above sequence returns to its original state. A single nucleotide Gx can be converted to A to provide a Hind site by the method described in US Patent Application No. 133,150 (to Bahl). This step exposes and alters the nucleotides to be converted and rearranges the sequence. This conversion does not change the next or last amino acid of the signal sequence. DNA binding is described by Hershfield et al.Proc.Natl.Acad.
Sci. USA (1974) 71, 3455-3459. Reverse transcriptase was developed by Bahl et al.
al.Proc.Natl.Acad.Sci.USA (1977) 74, 966−
970, as a DNA polymerase. Transformation was performed by Cohen et al.
alProc.Natl.Acad.Sci.USA (1973) 70, 3240−
Proceed as described by 3244. Example 5 Although the method described in Example 4 above is technically convenient, it concerns mature nuclear leader sequences and may only be useful for general development. The most useful research from a commercial standpoint is usually conducted on prokaryotic (bacterial) hosts. Although only a few such signal sequences are known for prokaryotic systems, one well-characterized sequence is that from TEM β-lactamases. (SutcliffeProc.Nath.
Acad. Sci USA (1976) 75, 3747-3741) This sequence is ideal for addition to a cloned gene except that there are no convenient restriction sites in place for precursor sequence processing. The closest restriction site to the signal sequence is the Mbo site, which attaches 16 foreign amino acids to the cloned gene product. However, the terminal part of the signal sequence, which is TTTGCT, can be removed by one of the techniques mentioned above.
Can be converted to TTTGAT. This latter array is
When read along the first few nucleotides of the TEM β-lactamase gene, Bcl
A restriction site exists for (TGATCA).
This changes the last amino acid from Ala (alanine) to Asp (aspartic acid), resulting in one foreign amino acid, Glu (glutamic acid) or His (histamine), depending on what the first added nucleotide of the foreign DNA is. acid) to the above gene product. To alter the nucleotide chain as described above, short fragments are synthesized using the methods described above. There is a restriction site (Tha) upstream of the above signal sequence, and a restriction site Mbo downstream. Further downstream there is a taq site. The limiting conditions for Tha are
Mc Connell et al., Nucleic AcidRes (1978)
5, 1729-1939, Mbo
The limiting conditions of Gelinas et al., J. Mol. Biol
(1977) 114:169–179,
The limiting conditions for Taq are as described by Sato et al., Proc. Natl.
The conditions described in Acad. Sci. USA (1977) 74; 542-546 are followed. However, regarding Mbo,
DNA is prepared in host cell GM119 lacking deoxyadenosine methylase to avoid methylation of regions and prevent Mbo cleavage. In this latter relationship, Marinus and Morris
See Mutat.Res. (1975) 28, 15-26.
Alternatively, the restriction endonuclease SAU
The isoschizome of 3A, Mbo , can be used to isolate DNA from the host. It will therefore be appreciated that this invention provides methods and vectors for the controlled accumulation of foreign cloned gene products.
Movement or transport of these products out of the host cell allows the products to be arrested and recovered with minimal restriction, eliminating the risk of degradation or destruction of either the host cell or the gene product itself. From the description of this specification, it will be apparent to those skilled in the art that various modifications can be made in addition to the invention described in this specification. Such variations are also considered to be within the scope of this invention. References related to the present invention are as follows. 1 Bahl, C.P., Wu, R., Stawinsky, J.and
Narang, SA, Proc.Nat.Acad.Sci., USA ,
(1977) 74 , 966-970. 2 Chang, A. and Cohen, S., Molec Gen.
Genet. , (1979) 168 , 111-115. 3 Chou, PYand Fasman, GD, Ann.Rev.
Biochem., (1978), 47 , 251-276. 4 Cohen, SNand Chang, ACY, Proc.
Nat.Acad.Sci., USA, (1972), 69 , 2110−
2114. 5 Cohen, SN, Chang, ACY, Boyer, H.
W.and Helling, R.B., Proc.Nat.Acad.Sci.,
USA, (1973) 70 , 3240:3244. 6 Ehrlich, SD, Proc.Nat.Acad.Sci., USA ,
(1977) 74 , 1680−1682. 7 Gelinas, RE, Myers, PA and Roberts,
RJ, J. Molec. Biol. , (1977) 114 , 169−179. 8 Gelfand, D., Shepard, H., O'Farrell, P.
and Polisky, B., Proc.Nat.Acad.Sci.,
USA, (1978) 75 , 5869−5873. 9 Grycazn, T.J., and Dubnau, D., Proc.
Nat.Acad.Sci., USA, (1978) 75 , 1428−
1432. 10 Hershfield, V., Boyer, H.W.
Yanofsky, C., Lovett, M.A. and Helsinki,
DR, Proc.Nat.Acad.Sci., USA , (1974)
71, 3455−3459. 11 Keggins, K.M., Lovett, PSand Duvall,
EJ, Proc.Nat.Acad.Sci., USA , (1978) 75 ,
1423−1427. 12 Kunst, F., Pascal, M., Lepesant−
Kejzlarova, J., Lepesant, J., Billault, A.
and Dedonder, R., Bio.Chemie. , (1974)
56, 1481−1490. 13 Kupersztoch, Y. and Helinski, D.,
Biochem.Biophys.Res.Commun., (1973) 54 ,
1451−1459. 14 Marinus, MGand Morris, NR, Mutat.
Res., (1975) 28 , 15-26. 15 Marmur, J., J. Molec. Biol. , (1961) 3 ,
208−218. 16 Maxam. A. and Gilbert, W., Proc. Nat.
Acad.Sci., USA, (1973) 73 , 3942−3946 17 McConnell, DJ, Searcy, DGand
Sutcliffe, J.G., Nucleic Acids Res. (1978)
5, 1729−1739. 18 Meadway, RJPh.D.Thesis, University
of Edinburgh, (1969). 19 Roberts, R.J., in DNA Insertion
Elements, Plasmi ds and Episomes,
(1977), Editors, Bukhari, AI, Shapiro,
J.A., and Adhya, S.L., Cold Spring.
Harbor Laboratory, Pages 757−468. 20 Ross, GWand O: Callaghan, C.H.
Meth.Enzymol., (1975) 43 , 69−85. 21 Sato, S., Hutchison, DAIII and
Harris, J.I., Proc.Nat.Acad.Sci.USA ,
(1977), 74 , 542−546. 22 Sharp, P. A., Sugden, B. and Sambrook,
J., Biochem. , (1973) 12 , 3055−3063 23 Sherratt, DJand Collins, JF, J.Gen.
Microbiol., (1973), 76 , 217−230. 24 Sutcliffe, JG, Proc.Nat.Acad.Sci.,
USA, (1978) 75 , 3737−3741.

[Brief explanation of drawings]

The accompanying drawings will be clear from the description below. FIG. 1 is an overall diagram showing a partial structural map of a DNA fragment containing the gene for β-lactamase obtained from Bacillus licheniformis; FIG.
FIG. 3 is an overall diagram showing the correspondence between many nucleotide sequences in the fragment shown in the figure and amino acid sequences present in the protein encoded by the fragment; FIG.
Figure 2 is a schematic diagram showing the typical appearance of a plate on which a strain is grown, after it has been subjected to a PVA assay to test β-lactamase activity. The symbol B in the figure indicates Bacillus subtilis. Figure 4 is an overall diagram illustrating the structures of E. coli plasmid POG2110, B. subtilis plasmid pOG1196 and bifunctional plasmid pOG2165; Figure 6 shows a typical appearance of a plate on which the bacterial strain grows after a PVA assay to test β-lactamase activity; Figure 6 shows B. licheniformis; This is a diagram of the nucleotides that constitute part of the Pen P gene.

Claims (1)

[Scope of Claims] 1. A cloning vector capable of autonomous replication in a Gram-positive host bacterium is created, the vector has a gene for a predetermined protein, and the predetermined protein is Rather than being native to the host bacterium, the gene is positioned and oriented in the vector to be controlled by an operator, promoter and ribosome binding site sequence, and the predetermined protein is controlled by an exogenous transport mechanism consisting of a portion of a Bacillus secretory leader sequence, by which the protein is secreted from the host bacterium, and the vector is introduced into the host bacterium.
A method for producing Gram-positive bacteria that can produce predetermined proteins by expression. 2 This is the part that creates a vector capable of autonomous replication, and the cloning vector is pC221,
pUB110, pC194, pUB112, pT127, pOG2165,
The method according to claim 1, comprising a portion obtained from a plasmid selected from the group consisting of pOG2110, pCS1006, pOG1196, pCS832 and plasmids derived therefrom. 3. The method according to claim 1, wherein said gene or said predetermined protein is controlled by a β-lactamase operator, a promoter, and a ribosome binding site base sequence. 4. The method according to claim 1, wherein the predetermined protein is a mammalian protein. 5. The method according to claim 1, wherein the predetermined protein is a mammalian hormone. 6. The method according to claim 1, wherein the predetermined protein is β-lactamase. 7. The method of claim 1, wherein the transport function is provided by a portion of the β-lactamase gene. 8. The method of claim 1, wherein the transport function is provided by a portion of a eukaryotic insulin signal peptide. 9 The above cloning vector contains restriction enzymes SStI,
2. The method of claim 1, having one specific site each for Hind, PstI and Bgl.