EP0303668A1 - Vecteurs d'expression stables - Google Patents

Vecteurs d'expression stables

Info

Publication number
EP0303668A1
EP0303668A1 EP19880902162 EP88902162A EP0303668A1 EP 0303668 A1 EP0303668 A1 EP 0303668A1 EP 19880902162 EP19880902162 EP 19880902162 EP 88902162 A EP88902162 A EP 88902162A EP 0303668 A1 EP0303668 A1 EP 0303668A1
Authority
EP
European Patent Office
Prior art keywords
plasmid
host cells
dna
vector
vectors
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19880902162
Other languages
German (de)
English (en)
Inventor
Andrew 4 St. Michaels Cottages Mountain
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UCB Celltech Ltd
Original Assignee
Celltech R&D Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celltech R&D Ltd filed Critical Celltech R&D Ltd
Publication of EP0303668A1 publication Critical patent/EP0303668A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • This invention relates to recombinant DNA technology and in particular to expression vectors for expression of foreign genes in microorganisms, especially gram positive microorganisms, and to methods by which such vectors may be obtained.
  • Bacillus subtilis The gram positive microorganisms of species Bacillus subtilis are potentially very useful for the cloning and expression of heterologous genes. This species has a significant advantage over the commonly used gram negative bacterial host, Escherichia coli, in that it is able to efficiently secrete many proteins into the medium, thus simplifying the extraction and purification of the desired protein. Additionally, B. subtilis is non-pathogenic with respect to humans. In view of these advantages a number of attempts have been made to develop recombinant DNA expression vector systems for use in B. subtilis.
  • subtilis plasmid have been constructed carrying a leu gene as marker (Tanaka and Sakaguchi (1978) Molec. Gen. Genet. 165 269-276). These leu + plasmids were further modified by the addition of an antibiotic resistance marker (Tanaka and Kawano (1980) Gene 10131-136). This particular line of research does not appear, however, to have resulted in the production of vectors suitable for cloning and efficiently expressing foreign genes. This apparent lack of success may be attributed to the use of an antibiotic resistance marker derived from B. subtilis i.e. the same source with which the plasmids are naturally associated and from which they were isolated.
  • Chromosomal integration is an extremely undesirable event if the aim is to produce vectors containing multiple copies of heterologous DNA sequences to obtain high level expression of foreign genes. If the plasmid becomes integrated into the chromosome the copy number of the heterologous DNA becomes that of the chromosome.
  • Staphylococcus aureus plasmids could replicate in B. subtilis led to investigations of the suitability of such plasmids as expression vectors for use in B. subtilis.
  • Use of such vectors to express cloned DNA in B. subtilis has been found to be associated with instability problems.
  • S. aureus replicons carrying cloned DNA are stably maintained in B. subtilis host cells only in the presence of selection pressure, e.g. antibiotics. This renders such vectors unsuitable for use in large scale industrial processes, in view of the requirement to use costly procedures to remove the antibiotic or other selection pressure.
  • the invention provides a vector suitable for cloning and expressing heterologous DNA in host cells of a given type comprising:
  • plasmid DNA derived from a plasmid which is normally stably maintained in said host cells
  • heterologous DNA comprising at least one restriction site which is unique in said heterologous DNA and which does not occur in said plasmid DNA, and DNA coding for at least one selectable marker;
  • said heterologous DNA is ligated within a non-unique restriction site of said plasmid and the vector is normally stably maintained in said host cells.
  • the invention also includes methods for obtaining vectors according to the first aspect.
  • the invention provides a method for obtaining vectors according to the first aspect of the invention comprising: providing (a) a plasmid which is normally stably maintained in host cells of a given type, and
  • vectors are "normally stably maintained” in the host cells and selected for stable maintenance if the vectors are present in substantially 100% of host cells after growth for at least 50 generations in the absence of selection
  • Partial digestion cleaves the plasmid at one or more of the non-unique restriction sites to give a plurality of. linear DNA molecules.
  • Ligation of the cassette of the heterologous DNA with these linear DNA molecules produces a number of different vectors since the cassette of heterologous DNA becomes inserted at or between the various non-unique restriction enzyme cleavage sites in the plasmid.
  • the vectors so produced are then selected for stability in the host cells. Stability is indicative that the cassette has been inserted at the optimum site(s) for insertion.
  • the stability of the vectors may be tested in small scale serial subculture experiments.
  • heterologous DNA for the cassette decreases the chance of recombination occurring and of the foreign gene becoming integrated into the chromosome.
  • heterologous' is used herein to indicate that the DNA is derived from a source(s) which differs from the species of microorganism used as the host for expression.
  • the selectable marker may be any gene conferring a phenotype which enables selection of transformants.
  • Commonly used selectable markers include genes coding for antibiotic resistance or sensitivity, or a characteristic selectable by the removal or addition of a specific metabolite.
  • the method of the invention is generally applicable and is particularly useful in the provision of vectors suitable for cloning and expressing genes in bacterial host cells especially in gram positive bacteria.
  • Particularly preferred bacterial host cells are members of the genus Bacillus, e.g. B. lichenformis , B. brevis, B. sphaericus, B. megaterium, B.polymyxa, B. circulans, and most especially in B.pumilis, B. subtilis and B. amyloliquefaciens hosts.
  • the plasmid used for preparation of the vectors is an endogenous plasmid, i.e. one which occurs or is found naturally in the host used for cloning and expression.
  • the plasmid may be a Bacillus plasmid, and is preferably a Bacillus pumilis. Bacillus amyloliquefaciens or a Bacillus subtilis plasmid. Examples of suitable plasmids are pLS11, pLS12 ⁇ LS13 , pLS14 as described by Tanaka et al (J. Bacteriol. 129, 1487 - 1494 (1977)) These plasmids are available from the Bacillus Genetic Stock Centre, at the Ohio State University, Ohio, USA.
  • the 8.5kb plasmid found in Bacillus strain 1E2 or substantially similar variants or mutants of the said plasmid are especially preferred.
  • This plasmid is believed to be identical to pLS14 and for convenience has been designated ⁇ POD2000 hereinafter in the present description.
  • a restriction map of pPOD2000 is provided hereinafter at Figure 1.
  • heterologous DNA of the vector additionally comprises at least one transcription terminator.
  • the transcription terminator may be derived from either Gram positive or Gram negative microorganisms.
  • the transcription terminator may be that of the B. amyloliquefaciens ⁇ - amylase gene.
  • transcription terminators derived from E. coli may be particularly suitable, such as the phage ⁇ to, or the rrnBT1T2 transcription terminator.
  • the vectors of the invention may be used for cloning and expressing foreign genes.
  • the foreign genes may be cloned into the vector using the at least one unique restriction enzyme recognition site present in the cassette of heterologous DNA.
  • additional unique restriction sites may be added to the cassette by adding a further li nker to the cassette to the at least one enzyme recognition site.
  • 'foreign gene' means a heterologous DNA sequence coding for a desired gene product.
  • the vector contains a suitable promoter, such as a heterologous promoter.
  • a suitable promoter such as a heterologous promoter.
  • the heterologous DNA of the vector of the invention may include a promoter suitably positioned in relation to the at least unique restriction to promote expression of a foreign gene inserted at the site.
  • the natural promoter of the foreign gene may be used and conveniently may be inserted into the vector with the foreign gene.
  • the promoter preferably allows high level expression of the desired foreign gene and does not destabilise the system resulting in plasmid loss.
  • the promoter further preferably does not allow high level expression during the growth phase of the culture of microorganism containing the hybrid plasmid.
  • the promoter may be a promoter expression from which is regulatable or otherwise controllable.
  • the natural promoter of the foreign gene may be used and, if desired, production of the RNA species may be made controllable by incorporating a regulating circuit such as an operator sequence and repressor gene into the system.
  • the promoter may be derived from a strain of Bacillus and if desired may be made controllable .
  • Suitable promoters, which may if desired be made controllable include, for example, subtilisin promoters, e.g. derived from B. amyloliquefaciens, amylase promoters e.g. derived from B. amyloliquefaciens, E. coli promoters e.g.
  • the invention further provides an expression vector for expression of a foreign gene product comprising a vector according to the first aspect of the invention having the foreign gene inserted at the at least one unique restriction site of the heterologous DNA of the said vector.
  • the invention also includes transformed host cells.
  • the invention further provides host cells of a given type transformed with a vector according to the first aspect of the invention or expression vector according to the third aspect of the invention.
  • the host cells are the endogenous host cells of the plasmid from which the vectors are derived.
  • the host cells are gram positive bacterial host cells, especially of the genus Bacillus, most especially of species B subtilis.
  • the cassette of heterologous DNA and the vectors of the invention may be prepared using methods well known in the recombinant DNA art.
  • the cassette comprising the desired heterologous DNA may be initially assembled conveniently on a plasmid.
  • the optimal plasmid for cassette assembly will vary depending on the required components of the cassette.
  • E. coli vectors containing multi-purpose cloning sites are particularly suitable for assembling the cassette prior feo insertion into the endogenous plasmid.
  • the overall size of the cassette may affect the stability of the vector. It is believed that it is advantageous to keep the cassette as small as possible, e.g. - a maximum size of less than about 2KB.
  • the selectable marker may be isolated as a restriction fragment and may be inserted at an appropriate point in the cassette. Where it is desired to add a transcription terminator to the cassette this must be positioned and oriented appropriately to prevent readthrough transcription from the foreign gene.
  • the ends of the cassette may be modified by adding linkers of the desired sequence thereby creating the desired restriction enzyme recognition sites.
  • a particularly preferred cassette is the 1.5KB HIND III cassette fragment as hereinafter described and shown in Figure 3.
  • the plasmid is partially digested and then purified prior to ligation with the cassette of heterologous DNA.
  • the linearised form of the plasmid i.e. the plasmid cleaved at only one of the multiple restriction sites, may be identified by size.
  • gel electrophoresis techniques may be used in which a complete digest of the plasmid using a restriction enzyme which cleaves the plasmid only once is run in an adjacent track and the linearised form of the plasmid is identified by its similarity of mobility. The linearised form is then purified and ligated with the cassette.
  • the host is a gram positive microorganism such as a Bacillus host this may be then transformed by, for example, a protoplast method. Where the host is a gram-negative microorganism transformation may be carried out using methods well known in the art. Transformants are selected using the selectable marker.
  • the desired promoter and/or the transcription terminator and foreign gene may be added to the cassette as one unit where for example the promoter and/or transcription terminator may be those naturally associated with the foreign gene.
  • heterologous promoter and foreign gene are cloned into an appropriate site of the cassette contained in the plasmid as described above. Transformants are selected and theirnability to express the foreign gene is examined. Stability of the transformants may then be investigated.
  • the foreign gene is added to the cassette and oriented in the vector such that transcription of the gene is in the direction of the transcription terminator.
  • vectors according to the present invention maintain cloned DNA with 100% stability for at least 70 generations.
  • Particularly preferred vectors according to the invention are ⁇ POD2152, pPOD2154, pPOD2158, pPOD2161, pPOD2167 and pPOD2168 as hereinafter specifically described.
  • Figure 1 shows a restriction map of plasmid pPOD2000
  • Figure 2 is a diagram showing plasmid restriction maps and a scheme indicating the mode of construction of plasmid pPOD2132
  • Figure 3 is a similar diagram indicating the mode of construction of plasmid pPOD2145
  • Figure 4 shows vector restriction maps of the various classes of vector arising from the insertion of the cassette
  • Figure 5 shows a restriction map of ⁇ POD2180
  • Figure 6 shows restriction maps of the various vectors resulting from the insertion of the ⁇ -amylase gene into the vectors of the invention. Description of the Specific Embodiments
  • Bacillus strain 1E2 was used as the source of the endogenous plasmid (a strain maintained by the Bacillus Genetic Stock Centre, The Ohio State University, 484 West, 12th Avenue, Columbus, Ohio 43210, USA). This strain was grown in Penassay broth (Difco Antibiotic Medium No. 3) and the cells harvested in late logarithmic growth phase (0.D600nm of 1.0). Plasmid DNA was then isolated from the cells by the method of Birnboim & Doly, 1979 (Nucl. Acid. Res. 7 : 1513). Agarose gel electrophoresis of such preparations revealed a single plasmid species 8.5kb in size, which was designated pPOD2000.
  • Plasmid copy number determinations were performed for pPOD2000 by the method of Projan et al, 1983 (Plasmid 9: 182-190) using 1E2 cells in early, mid and late logarithmic growth phase and at several time points in stationary phase. The plasmid was found to maintain a copy number of 10 per chromosome irrespective of growth phase.
  • a restriction map of pPOD2000 was generated by standard techniques, (described in Maniatis et al, 1982 - Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
  • the plasmid contains five HindIII sites distributed fairly evenly around the map. A cassette was therefore constructed with Hindlll ends suitable for insertion into pPOD2000 in five different positions. pPOD2000 contains no recognition sites for the enzymes BamHl and Sma1, and the cassette was constructed with unique sites for these two enzymes to facilitate the cloning of foreign genes as described below.
  • Example 2
  • pUC8 can be purchased from Bethesda Research Laboratories, Cambridge, UK.
  • Staphylococcus aureus plasmid pC194 (lordanescu, S et al 1978, Plasmid 1: 468-479) was chosen as the source of the antibiotic resistance marker for three reasons: (a) because its chloramphenicol resistance gene is known to be suitable for selection of B. subtilis transformants using the protoplast transformation procedure (Chang and Cohen, 1979, Molec. Gen. Genet. 168: 111); (b) because the promoter of that gene is relatively inefficient in B. subtilis (A. Mountain & M. Nugent, unpublished) and therefore unlikely to destabilise plasmids in that host by readthrough transcription or metabolic load effects and (c) because pC194 has been fully sequenced and partially characterised
  • JM101 ATCC strain 338766 with selection for ampicillin resistant transformants.
  • Several transformants were identified which were also resistant to chloramphenicol. Among these one was identified by restriction analysis in which the Cm r gene was oriented with its
  • This plasmid was designated pPOD2103.
  • plasmid pPOD2103 0.5 ⁇ g was digested with BamHl and treated with calf intestinal phosphatase, followed by phenol extraction and ethanol precipitation. Following resuspension in TE the digest was ligated with 0.1 ⁇ g of the 189bp ⁇ to fragment. The ligation mixture was used to transform competent cells of E. coli strain DH1 (ATCCH-strain 33849), with selection for ampicillin resistant transformants. Plasmid minipreparations were performed on 12 such transformants by the method of Birnboim & Doly, 1979 (Nucl. Acid. Res. 7: 1513).
  • BamHl site 1, Figure 1 As a unique site to facilitate further cloning it was necessary to remove the other BamHl site, adjacent to the Smal site (BamHl site 2, Figure 1). This was accomplished by using the DNA polymerising activity of the Klenow fragment of E. coli DNA polymerase to fill in the recessed 3' ends of a partial
  • Recessed 3' ends in the digest were then filled in by using the Klenow fragment of DNA polymerase in the presence of all four deoxyribonucleotide triphosphates as described in Maniatis et al, 1982 (Molecular Cloning, A Laboratory Manual). Following a further phenol extraction and ethanol precipitation the digest was resuspended in TE buffer and electrophoresed on a preparative agarose gel. The linear form was purified from the gel by electroelution and phenol extraction. Following ethanol precipitation the approximately 0.2 ⁇ g of linear pPOD2104 recovered was resuspended in 10 ⁇ l of TE buffer and ligated in a volume of 100 ⁇ l to effect circularisation. The ligation mixture was transformed into E .
  • Hindlll digest was then subjected to preparative agarose gel electrophoresis and the 1.5 kb Hindlll fragment containing the Cm r gene and ⁇ to was purified by electroelution and phenol extraction, and resuspended in TE buffer.
  • 1 ⁇ g of pBR322 DNA (Bolivar et al, 1977, Gene 2: 95-113) was digested to completion with HindIII and treated with calf intestinal phosphatase. Following phenol extraction, ethanol precipitation and resuspension in TE, 0.1 ⁇ g of this DNA was ligated with 0.1 ⁇ g of the 1.5kb HindIII fragment. The ligation mixture was transformed into competent cells of E.
  • coli strain DH1 with selection for ampicillin resistant transformants. Among these several were identified which also displayed resistance to chloramphenicol up to 20 ⁇ g per ml. Complete single digests with HindIII, EcoRl, Clal and BamHl, and complete double digests with BamHl/Smal and BamHl/EcoRl were performed on plasmid preparations of two of these clones. Agarose gel electrophoresis of these digests showed all were consistent with both plasmids comprising pBR322 carrying a single insert of the 1.5kb HindIII fragment in the HindIII site. One of the two hybrid plasmids was designated pPOD2145 (see Figure 3). A restriction map of the 1.5kb HindIII fragment, hereafter referred to as the HindIII cassette fragment, is given in Figure 3.
  • Partial HindIII digests were performed on pPOD2000 by incubating 5 ⁇ g of pPOD2000 DNA at 37oC with 1 unit of HindIII for 5, 10 and 20 minutes respectively. 1/10th of each digest was subjected to agarose gel electrophoresis and the remaining 9/10this stored at -20oC. The 10 minute digest was adjudged to be the most suitable since it contained the greatest proportion of the required partial digestion product, i.e. pPOD2000 linearised by cleavage at only one of the five HindIII sites, the linearised form being identified by electrophoresing on the same gel a complete Sall digest of pPOD2000. (As indicated in Figure 1, pPOD2000 contains only a single Sail site).
  • the remaining 9/10 of the 10 minute partial HindIII digest was phenol extracted and ethanol precipitated. Following resuspension in TE the digest was treated with calf intestinal phosphatase, then electrophoresed on a preparative agarose gel. The linearised form was purified from the gal by electroelution and phenol extraction. Following ethanol precipitation the DNA was resuspended in TE.
  • the number of colonies resistant to the antibiotic was expressed as a percentage of the number growing on the L-agar plate, and taken as representing the proportion of the population carrying the plasmid.
  • the results are given in Table 2. They show that insertion of the cassette in either orientation into pPOD2000 at HindIII sites 1 or 4, or replacing the smallest pPOD2000 HindIII fragment between sites 1 and 2, gives hybrid plasmids which are maintained with 100% stability in the absence of selection. In the same experiment pC194, the Staphyloccoccus aureus plasmid from which is taken the Cm r gene contained in the cassette, is maintained with only 90% stability. Table 2
  • the usefulness of the vectors derived from pPOD2000 and described in Example 4 was examined by cloning into one representative of four (out of the seven) classes an ⁇ -amylase gene originating in a Bacillus species closely related to B. subtilis.
  • the ⁇ -amylase gene was subcloned from a hybrid plasmid designated pPOD2180 which comprised the vector pBD64 (Gryczan et al., 1980, J. Bacteriol. 141: 246-253) with a 2.5kb partial Sau3A fragment of the closely related Bacillus species cloned into the BglII site.
  • a restriction map of pPOD2180 is given in Figure 5.
  • the ⁇ -amylase gene is efficiently transcribed and translated in B. subtilis using the natural gene expression signals of that gene.
  • 2 ⁇ g of ⁇ POD2180 DNA was digested to completion with first Bglll, then BamHl, and the digest electrophoresed on a preparative agarose gel.
  • the 2kb fragment which contains the entire ⁇ -amylase gene was purified from the gel by electroelution and phenol extraction. Following ethanol precipitation the fragment was resuspended in TE.
  • 1 ⁇ g of each of the plasmids pPOD2152, pPOD2158, pPOD2168, and pPOD2154 was digested to completion with BamHl, and treated with calf intestinal phosphatase. Following phenol extraction and ethanol precipitation the digests were resuspended in TE.
  • Plasmid preparations were performed on several transformants resulting from ligation of the 2kb fragment carrying the amylase gene with each of the vectors derived from pPOD2000.
  • a series of restriction digests were performed on these plasmids, with results which confirmed that they comprised the intact vectors with the intact 2kb insert carrying the amylase gene.
  • the digests also showed that for each of the four vectors hybrids were recovered in which the cloned 2kb fragment was oriented such that the amylase gene was transcribed towards the ⁇ to terminator.
  • hybrids were also recovered in which the 2kb fragment was in the opposite orientation, i.e.
  • HB medium permits much higher biomass to be achieved for strain 1A289 than L-broth or other conventional complex media, and it was therefore used in the stability tests to prolong the growth phase.
  • the number of colonies resistant to the antibiotic was expressed as a percentage of the number growing on the L-agar plate, and taken as representing the proportion of the population carrying the plasmid.
  • plasmids pPOD2180, 2191, 2192, 2193, 2194, 2195 and 2196 the 100 colonies were also picked onto L-agar containing 1% starch to test for retention and expression of the cloned amylase gene: for each plasmid a 100% correlation was observed between growth on chloramphenicol and production of clearing zones on plates containing starch, suggesting the results were not complicated by structural instabilities.
  • Table 3 The results are presented in Table 3.
  • This strain contains a mutation, sacU h , which leads to increased production of the B. subtilis ⁇ -amylase, levansucrase and proteases, and also to increased instability of a number of hybrid plasmids, including a high copy number hybrid comprising the ⁇ -amylase gene of B.
  • subtilisin from B. amyloliquefaciens.
  • the gene was taken from plasmid pPT30, which comprises a 3.4Kb EcoRl fragment containing the entire subtilisin gene cloned into pUB110 (Thomas et al, 1985, Nature 318:375-376). 3ug of pPT30 DNA was digested with EcoRl, electrophoresed on an agarose gel and the 3.4Kb EcoRl fragment containing the subtilisin gene purified as described in Example 2a.
  • Example 2c The recessed 3' ends of this fragment were filled in as described in Example 2c.
  • lug of pPOD2152 was digested with BamHl and the recessed 3' ends filled in as described in Example 2c.
  • 0.5ug of this pPOD2152 DNA was ligated with 0.5ug of the subtilisin fragment and the mixture transformed into B. subtilis strain 1A289 as described in Example 3.
  • Colonies carrying the subtilisin gene cloned into pPOD2152 were identified by their production of a large clearing zone on L-agar plates containing chloramphenicol and 1% skimmed milk.
  • subtilisin The presence of the cloned subtilisin fragment in these clones, and the orientation of the subtilisin gene with respect to the ⁇ to transcription terminator was established by perfroming restriction digests on purified plasmid DNA. Similarly the same fragment carrying the subtilisin gene was cloned into the BamHl site of the Staphylococcus aureus plasmid pUB110, the replicon from which cloning vectors and expression vectors for use in B. subtilis are most commonly derived (Gryczan, 1982, in "The Molecular Biology of the Bacilli", Ed. D.A. Dubnau, Academic Press, pp 307-329).
  • Segregational stability tests were then performed on representative clones as described in Example 6, except that the presence or absence of the hybrid plasmids carrying the subtilisin gene was scored both by antibiotic restistance (chloramphenicol for pPOD2152 hybrids, and kanamycin for pUB110 hybrids) and the presence or absence of large clearing zones on L-agar plates containing 1% skimmed milk.
  • pPOD2380 was chosen as a representative pP0D2152 clone in which the subtilisin gene was oriented such that it was transcribed from its own promoter towards ⁇ to, and was identical to pPOD2191 shown in Figure 6 except with the amylase gene replaced by the sutilisin gene.
  • pPOD2390 and pPOD2391 were chosen as representative pUB110 clones with the subtilisin gene in opposite orentations in the BamHl site. The results are presented in Table 4. and show that pPOD2380 was maintained with 100% stability after 50 generations in the absence of selection, while the pUB110 hybrids proved segregationally unstable.
  • a 2Kb BamHl fragment carrying this amylase/ ⁇ -lactamase fusion - expressing ⁇ -lactamase using the amylase promoter, translation initiation region and signal sequence coding region - was ligated with pPOD2152 digested with BamHl, and the mixture transformed into 1A289 protoplasts. Chloramphenicol resistant transformants were screened for ampicillin resistance to identify hybrids carrying the amylase/ ⁇ -lactamase fusion fragment.
  • Plasmid pPOD2395 was identified by restiction digests as a hybrid comprising pPOD2152 carrying the 2Kb amylase/ ⁇ -lactmase fusion oriented such that transcription of the ⁇ -lactamase gene from the amylase promoter was towards ⁇ to. Segregational stability tests were performed on 1A289 carrying pPOD2395 and pPOD2152 as described in Example 6, except that presence or absence of the plasmids was scored by resistance to both chloramphenicol and ampicillin for pPOD2395, and to chloramphenicol only for pPOD2152. Both plasmids proved to be maintained with 100% stability after 70 generations in the absence of selection.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measurement And Recording Of Electrical Phenomena And Electrical Characteristics Of The Living Body (AREA)
  • Oscillators With Electromechanical Resonators (AREA)
  • Complex Calculations (AREA)

Abstract

Le vecteur sert au clonage et à l'expression d'ADN hétérologue dans des cellules hôtes d'un type donné (en particulier gram positif, y compris des cellules hôtes Bacillus et notamment B.subtilis, B.pumilis ou B.amyloliquefaciens). Un procédé permettant d'obtenir ce type de vecteur est également décrit. Lesdits vecteurs sont obtenus à partir de plasmides qui sont normalement conservés à l'état stable dans les cellules hôtes données par clonage d'une cassette d'ADN hétérologue comprenant un marqueur sélectionnable, au moins un site de restriction unique qui ne se trouve pas dans le plasmide et éventuellement une terminaison de transcription et un promoteur dans un site non unique du plasmide, suivi par une sélection des vecteurs résultants pour la conservation stable dans les cellules hôtes données. Cette approche augmente au maximum les chances d'obtenir un vecteur stable ainsi que la possibilité de trouver une position sur le plasmide où l'insertion d'ADN n'interrompt pas une région essentielle ou importante pour la réplication et où la conservation stable est grandement accrue. La présente invention décrit des vecteurs de Bacillus spécifiques (pPOD2152, pPOD2154, pPOD2158, pPOD2161, pPOD2167 et pPOD2168), dont on a découvert qu'ils conservent l'ADN hétérologue cloné avec une stabilité de 100 % pendant au moins 70 générations.
EP19880902162 1987-03-03 1988-03-03 Vecteurs d'expression stables Withdrawn EP0303668A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB878704908A GB8704908D0 (en) 1987-03-03 1987-03-03 Vectors
GB8704908 1987-03-03

Publications (1)

Publication Number Publication Date
EP0303668A1 true EP0303668A1 (fr) 1989-02-22

Family

ID=10613222

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19880902162 Withdrawn EP0303668A1 (fr) 1987-03-03 1988-03-03 Vecteurs d'expression stables

Country Status (6)

Country Link
EP (1) EP0303668A1 (fr)
JP (1) JPH01502795A (fr)
AU (1) AU615179B2 (fr)
FI (1) FI885060A0 (fr)
GB (1) GB8704908D0 (fr)
WO (1) WO1988006622A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6027388A (ja) * 1983-07-22 1985-02-12 Green Cross Corp:The プラスミツド

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8806622A1 *

Also Published As

Publication number Publication date
AU1365088A (en) 1988-09-26
FI885060A (fi) 1988-11-02
JPH01502795A (ja) 1989-09-28
AU615179B2 (en) 1991-09-26
GB8704908D0 (en) 1987-04-08
FI885060A0 (fi) 1988-11-02
WO1988006622A1 (fr) 1988-09-07

Similar Documents

Publication Publication Date Title
DE3856339T2 (de) Transformation von alkalophilen Bazillus-Stämmen
EP0283075B1 (fr) Clonage moléculaire et expression de gènes codant pour des enzymes protéolytiques
EP0284126B1 (fr) Amplification de gènes stable dans l'ADN chromosomique des prokaryotes
AU570709B2 (en) Cloning and expression in industrial species
US4666848A (en) Polypeptide expression using a portable temperature sensitive control cassette with a positive retroregulatory element
EP0127328A2 (fr) L'emploi d'une intégration dans des chromosomes pour stabiliser des gènes hétérologues
US4910141A (en) 3'-expression enhancing fragments and method
US5017477A (en) Enhancing DNA sequencies derived from the sacQ gene
US5084383A (en) Bacillus subtilis strain whose extracellular protease activities are reduced, method for obtaining the strain and method for secreting proteins by using the strain
AU615179B2 (en) Stable expression vectors
CA1201669A (fr) Methode de genie genetique pour l'obtention de plasmides pour transformer des microorganismes gram positifs
JP2558249B2 (ja) 正のレトロレギユレーシヨン因子
Jung et al. Integration and amplification of the Bacillus sp. 79-23 cellulase gene in the Bacillus subtilis 168 chromosome
Aiba et al. Production of α-Amylase by Bacillus Stearothermophilus (pAT9) and Gene Manipulation to Improve the Stability of the Recombinant Plasmid

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19881027

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17Q First examination report despatched

Effective date: 19911016

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19920227