EP0370103A1 - Bacteries gram-positives deficientes en proteases et leur utilisation comme organismes hotes pour la production de produits recombinants - Google Patents

Bacteries gram-positives deficientes en proteases et leur utilisation comme organismes hotes pour la production de produits recombinants

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Publication number
EP0370103A1
EP0370103A1 EP89906977A EP89906977A EP0370103A1 EP 0370103 A1 EP0370103 A1 EP 0370103A1 EP 89906977 A EP89906977 A EP 89906977A EP 89906977 A EP89906977 A EP 89906977A EP 0370103 A1 EP0370103 A1 EP 0370103A1
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EP
European Patent Office
Prior art keywords
protease
residual
rsp
lysostaphin
rcp
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Withdrawn
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EP89906977A
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German (de)
English (en)
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EP0370103A4 (en
Inventor
Peter Blackburn
Michael Arthur Lonetto
Edward L. Chang
June Polak
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Applied Microbiology Inc
Original Assignee
NEW YORK HEALTH RES INST
Applied Microbiology Inc
Health Research Institute
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Publication of EP0370103A1 publication Critical patent/EP0370103A1/fr
Publication of EP0370103A4 publication Critical patent/EP0370103A4/en
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)

Definitions

  • This invention relates to strains of gram- positive bacteria such as Bacillus subtilis which are protease-deficient to methods and tools useful in the isolation of such strains and to the use of these strains for the production of heterologous proteins.
  • Bacillus and other gram-positive organisms will, on the other hand, secrete proteins into the extracellular culture medium. This is advantageous from the standpoint of purification of heterologous proteins.
  • bacteria such as Bacillus are more cost effective in production of proteins than competing hosts, such as yeast and mammalian cell cultures due to higher growth rates, the use of less expensive growth media, and generally more facile cultivation procedures.
  • Bacillus is a fermentation organism grown routinely on an industrial scale, and one species, B. subtilis, is the most widely studied gram-positive organism. It is non-pathogenic, and unlike E. coli, it does not produce endotoxin, clearly desirable features when the recombinant products are destined for medical or veterinary use.
  • Plasmid cloning vectors have been constructed which make it possible to introduce and express foreign genes in Bacillus hosts. In general, these cloning vectors are secretion vectors, in which the signal sequence of a secretory Bacillus protein is fused to the structural gene coding for the desired heterologous protein.
  • This construct is then cloned into a plasmid capable of replicating in Bacillus.
  • These plasmids have unique restriction sites, and encode selectable antibiotic resistance markers useful in isolation of transformant organisms.
  • the two major Bacillus extracellular proteases are the neutral protease (NP), a metalloenzyme sensitive to ethylenediaminetetraacetic acid (EDTA), and the alkaline protease (AP), a serine protease having optimum activity at alkaline pH.
  • the structural genes for these two major proteases have been cloned and used to produce in vitro derived defined deletions which inactivate the respective protease genes.
  • Stahl et al. 158 J. Bacteriol. 411 (1984); Yang et al., 160 J. Bacteriol. 15 (1984); Kawamura et al., 160 J. Bacteriol. 442 (1984).
  • mutant strains with deletions in the structural genes for the two major proteases are referred to herein as aprE- /nprE- double mutants.
  • Mutant strains which are phenotypically deficient in alkaline and neutral protease, but in which the nature of the mutation is not specified are referred to herein as AP-/NP- double mutants.
  • FIG. 1 shows growth of wild-type and protease-deficient mutants of B. subtilis BD170 on TBAB agar containing 1% skim milk at 37°C for 16h are shown. Clear zones surrounding the colonies are due to proteolytic degradation of casein.
  • AP-/NP- strains of B. subtilis exhibit little or no activity toward casein in this assay as shown in Fig. 1. Nevertheless, AP- /NP- strains of B. subtilis still degrade many recombinant secretory proteins of interest. For example, AP-/NP- strains of B.
  • subtilis degrade heterologous proteins such as lysostaphin, prolysostaphin, micrococcal nuclease, and fusion proteins comprising eukaryotic proteins, including interleukin-1 and tissue-plasminogen activator, fused with the secretory and proenzyme sequences of prolysostaphin to enable their secretion from the bacterium.
  • heterologous proteins such as lysostaphin, prolysostaphin, micrococcal nuclease, and fusion proteins comprising eukaryotic proteins, including interleukin-1 and tissue-plasminogen activator, fused with the secretory and proenzyme sequences of prolysostaphin to enable their secretion from the bacterium.
  • RSP residual serine protease
  • RCP sulfhydryl-dependent residual cysteine protease
  • strains of B. subtilis can be developed and identified which are deficient in not only the neutral and alkaline proteases, but also in one or both of the residual proteases.
  • Such strains are highly suitable for use as host organisms for the production of heterologous proteins which would be susceptible to the proteolytic activity of the residual proteases.
  • Fig. 1 shows a casein-agar plate demonstrating the protease activity of wild type B. subtilis BD170, and its AP-/NP- and AP-/NP- mutants;
  • Fig. 2 shows the halflife of a recombinant protein, lysostaphin, throughout growth in cultures of B. subtilis BD170 wild type, B. subtilis BD170 AP- /NP-, and the protease-deficient isolate B. sphaericus 00;
  • Fig. 3 shows the chromatographic separation of the residual cysteine protease (RCP) and the residual serine protease (RSP);
  • Fig. 4 shows the respective activities of RCP and RSP toward specific polypeptide substrates, prolysostaphin and lysostaphin;
  • Fig. 5 shows the effect of inhibition of RCP and RSP on the stability of a heterologous secretory protein
  • Fig. 6 shows the N-terminal amino acid sequence of RSP
  • Fig. 7 shows the preferred oligonucleotide probe sequence for RSP. Detailed Description of the Invention
  • RSP residual serine protease
  • RCP sulfhydryl-dependent residual cysteine protease
  • Figure 2 shows lysostaphin inactivation as a function of growth stage comparing B. subtilis BD170 wild-type (W), with B. subtilis AP-/NP- (D), and
  • lysostaphin produced by recombinant DNA technology as described in International Patent Application Serial No. PCT/US87/00873 published as WO87/06264 on October 22, 1987 and incorporated herein by reference
  • This key observation illustrates that the instability of this secretory recombinant product is largely effected by the activity of the residual proteases, residual cysteine protease (RCP) and residual serine protease (RSP) in those cultures.
  • the chromatographic conditions were by anion exchange HPLC on a column of Mono Q (Pharmacia) using the following conditions: Solvent A: water, Solvent B: 100 mM Na 2 HPO 4 , pH 7.0, 1.0M in NaCl . Gradient: 5-100% solvent B in 15 minutes at 1.0 ml per minute. Peaks were detected on-line by A 210 and A 280 . The positions of the arrows indicate the beginning and end of the salt gradient.
  • RSP can degrade both of the heterologous proteins prolysostaphin and lysostaphin (Fig. 4).
  • RCP has no effect on lysostaphin, but can convert prolysostaphin to lysostaphin.
  • Prolysostaphin is inactive and the conversion of the heterologous protein prolysostaphin to enzymically active lysostaphin forms the basis for an assay for RCP.
  • the inactivation that accompanies the degradation of the heterologous protein lysostaphin forms the basis of an assay for RSP.
  • the unmodified host is maintained in the culture collection of the Public Health Research Institute, New York, New York. This host is of importance nob only because it is but in that it can be transformed by plasmids pBC16-1L and pROJ6499-1L to produce and process prolysostaphin to lysostaphin despite the absence of AP, NP, RSP, and RCP protease activities.
  • mutant strains deficient in either or both of the residual proteases can be selected from mutant populations created by any known method.
  • Mutant strains are strains of microorganisms having one or more mutations, i.e. changes, in the genetic sequence of the organism. These mutations can be caused by any of a variety of mutagenic methods including exposure to radiation or chemical mutagens, transposition, recombination and restriction enzyme digestion. The resulting mutant strain can differ from the parent strain due to base-pair replacement, i.e.
  • deletion mutants are preferred because of the possibility of reversion in base-pair replacement or insertion mutants.
  • Such deletion mutants are preferably formed by known methods for specific in vitro deletions in the appropriate gene.
  • the identity of the residual protease it is also possible to eliminate the effect of the residual RCP and RSP produced by a non-mutant microorganism by chemically inhibiting or inactivating the effect of the RCP and RSP as they are produced in the culture medium by addition of chemicals for example pHMB, PMSF, and peptide analogue inhibitors such as antipain and metal chelators such as EDTA.
  • the first step in creating in vitro site specific deletion mutants deficient in RSP and/or RCP activity requires that the respective genes coding for these proteases within the bacterial genome be cloned. Toward this end, the N-terminal amino acid sequence of RSP has been determined, as shown in
  • Fig. 6 Based on the amino acid sequence of the protein, regions of least ambiguous DNA sequence, as predicted from codon degeneracy, were identified. Oligonucleotide probes corresponding to these amino acid sequences can be synthesized for use in hybridization studies to identify restriction fragments containing the residual protease gene. As appropriate, the codon degeneracy is accommodated by the use of mixed base oligonucleotide probes. For example, three such oligonucleotide mixed probes appropriate for regions of the N-terminal amino acid sequence generated for RSP are shown in Fig. 7. Other probe sequences based on other parts of the protease amino acid sequence can also be used.
  • the genes encoding RSP and RCP can be located by hybridizing the 32 P-labeled oligonucleotide probes with restriction digests of Bacillus chromosomal DNA.
  • the specific fragments which are identified as containing the RSP or RCP gene then are cloned in a plasmid and can be sequenced or subjected to site specific mutagenesis preferably to create deletion mutants.
  • antibiotic resistance markers can be inserted into the deleted gene.
  • the protease genes in B. subtilis are then inactivated by homologous recombination and mutants are selected by growth in the presence of antibiotic and tested for residual protease activity.
  • AP-/NP-, stationary phase culture supernatants were fractionated by ion-exchange high performance liquid chromatography (HPLC) on Mono Q (Pharmacia).
  • Solvent B 100 mM Na 2 HPO 4 , pH 7.0, 1.0M in NaCl.
  • Gradient 5-100% solvent B in 15 minutes at 1.0 ml per minute. Peaks were detected on-line by A 210 and A 280 .
  • 1.0 mg/ml solution of lysostaphin is incubated with an equal volume of culture supernatant (or other sample containing the RSP enzyme) forms the basis for the quantitative assay although other suitable heterologous proteins could be used as well. Shorter or longer time periods could be used, so long as the reaction is allowed to proceed for sufficient time to allow degradation of at least a part of the lysostaphin or other heterologous proteins if RSP is present.
  • One unit of protease activity in this assay is the amount of protease that produces 50% inactivation of the lysostaphin incubated as described.
  • the residual activity of the lysostaphin was measured turbidometrically by its activity against a cell suspension of heat killed Staphylococcus aureus and was compared with an untreated lysostaphin control.
  • RCP can be quantitatively estimated under similar conditions with 1 unit being defined as that amount of RCP bringing about the 50% activation of prolysostaphin to lysostaphin. Since RSP will degrade lysostaphin produced from prolysostaphin by RCP, however, the assay for RCP in culture supernatants that also contain RSP requires that RSP be first inactivated by incubation with phenylmethysulfonyl fluoride (PMSF) or some other RSP inhibitor. In the presence of PMSF, the RCP activity of B. subtilis AP-/NP- cultures will convert prolysostaphin to lysostaphin and permit accumulation of the enzyme.
  • PMSF phenylmethysulfonyl fluoride
  • a convenient assay for RCP and RSP can be performed by analysis of heterologous proteins such as prolysostaphin and lysostaphin degradation and accumulation of degradation products by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 4) Furthermore, the activity of RCP and RSP, respectively, can be quantitated by their activities towards heterologous proteins such as prolysostaphin and lysostaphin substrates labeled radioactively with 125I or with fluorescein isothiocyanate.
  • the degradation of the labeled heterologous proteins, or accumulation of degradation products, can be conveniently measured after acid precipitation to separate labeled products from labeled substrate and the measurement of either acid- soluble label or acid-insoluble label, respectively.
  • RSP was found to be inactivated by PMSF.
  • RSP is inhibited by the protease inhibitor antipain (Phe-Arg-Val-Arg-Al).
  • RCP is unaffected by PMSF, but is inactivated by the sulfhydryl reactive agent, p-hydroxymercuribenzoate (pHMB).
  • pHMB p-hydroxymercuribenzoate
  • FIG. 5 shows the production of interleukin 1-micrococcal nuclease fusion proteins by cultures of the double proteaseminus strain B. subtilis BD170 AP-/NP-.
  • VY Veal/Yeast
  • Protease inhibitors were added when the cultures reached 220 Klett units and again at the time of harvest.
  • Blots were incubated with rabbit antibodies to micrococcal nuclease (top) or rabbit antibodies to interleukin 1 (bottom) and detected with goat anti-rabbit IgG-alkaline phosphatase conjugate.
  • the interleukin 1 standard was run in lane X.
  • N-terminal amino acid sequence of the purified RSP isolated above was determined by Edman degradation with an automated gas-phase protein sequencer (Applied Biosystems).
  • the protein purified by ion-exchange HPLC was desalted by reverse phase chromatography on C 3 columns eluted with a linear gradient of 0-75% (v/v) acetonitrile in 0.1% trifluoroacetic acid (TFA).
  • FFA trifluoroacetic acid
  • Autocatalytic degradation of RSP and RCP was prevented by inactivation of the enzymes by reaction with PMSF and pHMB, respectively.
  • Amino acid analysis was performed on acid hydrolysates of the purified RSP protein.
  • N-terminal amino acid sequencing was performed on the intact protein RSP.
  • N-terminal amino acid sequence of RSP was determined to be as shown in Fig. 6. Based on this amino acid sequence data, regions of least ambiguous DNA sequence, as predicted from code degeneracy, were identified and oligonucleotide probes corresponding to these sequences were synthesized for use in hybridization studies to locate the residual serine protease gene. Where necessary, code degeneracy was accommodated by synthesis of mixed base probes.
  • Suitable oligonucleotide probes are shown in Fig. 7. Of course, other probe sequences based upon other parts of the protease amino acid sequence could also be effectively used.
  • peptide fragments of the respective proteins are separated especially by reverse phase chromatography on C 18 columns eluted with similar solvents (i.e. acetonitrile or iso-propanol in 0.1% TFA).
  • Appropriate fragmentations of the proteins to component peptides are made by CNBr cleavage. Fragmentation is most readily accomplished enzymically by digestions with trypsin after Lys and Arg residues, or with clostripain after Arg residues. Clostripain digestion is particularly appropriate for RSP since it has only 4 Arg residues per molecule. Other known specific cleavage methods may also be useful for generating specific peptide fragments of RSP and RCP. Restriction digests of B.
  • subtilis IS75 or BD170 chromosomal DNA can be prepared using HindIII and Sau3A1 restriction nucleases.
  • Appropriately sized (approximately 3kb) RCP and RSP specific fragments resulting from the several different digests can be isolated from preparative agarose gels or from sucrose density gradients (5-20%) after non-equilibrium centrifugation at neutral pH.
  • Plasmid libraries can be constructed that contain the isolated RSP and RCP specific fragments using pUC or pBR322 plasmid vectors cut at HindIII or Bam HI sites and cloning in E. coli.
  • Plasmid DNA is prepared from positive clones and characterized by restriction analysis. For example, these restriction digests can be examined by Southern analysis using the 32 P- labelled oligonucleotide probes for RSP and RCP specific sequences. Fragments hybridizing with the
  • protease-specific restriction fragments With protease-specific restriction fragments in hand, inactivation of their respective protease genes can be accomplished by homologous recombination.
  • an antibiotic resistance marker can be advantageously inserted into the deleted gene in the plasmid.
  • genes encoding kanamycin, erythromycin, fusidic acid, and chloramphenicol resistance can be inserted into the deleted genes for AP, NP, RSP and RCP, respectively.
  • the vector encoding for the mutated protease sequence and the respective antibiotic resistance marker are then used to transform B. subtilis.
  • mutant strains multiply protease deficient, e.g. triple mutant strains having the phenotype AP- /NP-/RSP- and AP-/NP-/RCP- and quadruple mutant strains having the phenotype AP-/NP-/ RSP-/RCP-, can be selected.
  • EcoRI-digested B EcoRI-digested B.
  • a limitation of this approach to systematically inactivate the protease genes is that the antibiotic resistance markers remain in the host genome. In the case of AP-/NP-/RCP-/RSP- mutants this entails the use of four separate antibiotic resistance markers. In turn, because of this limiting antibiotic resistance of tire host, the range of useful cloning vectors is restricted to those that encode additional antibiotic resistance. Thus, ideally the resistance markers in the genome should be inactivated. Several approaches can be used to achieve this goal.
  • the inactivation of the protease genes could be achieved by congression and thus avoid the insertion of the antibiotic resistance marker.
  • Lysostaphin activity can be detected by the appearance of clear halos in an agar overlay containing a suspension of live or heat killed S. aureus.
  • inactivation of lysostaphin and the activation of prolysostaphin to lysostaphin can be visualized in order to screen colonies to specifically detect RSP and RCP, or their inactivation, by the appearance or lack of lysostaphin-dependent halos, respectively.
  • Tn917 encoding erythromycin resistance
  • Tn917 can be ligated into the disrupted protease gene in pUC8, and used to inactive the host protease genes by homologous recombination. Recombinants can be selected by growth in the presence of erythromycin.
  • This approach has the advantage that the transposable element can be lost from the genome when grown subsequently in the absence of erythromycin selection. This can be confirmed by sensitivity to erythromycin.
  • Another approach is to ligate an intact fusidic acid resistance marker to a fragment carrying a truncated chloramphenicol resistance marker (encoding the first 2/3 of the chloramphenicol acetyl transferase (cat) gene).
  • the hybrid construct encoding fusidic acid resistance would be inserted into the deleted protease gene in the pUC vector. This construct would be used as described above to insertionally inactivate after homologous recombination the respective protease genes for RSP and RCP by selection for fusidic acid resistance.
  • the fusidic acid marker can likewise be inactivated by homologous recombination.
  • the erythromycin resistant plasmid pRN5101 encodes a temperature sensitive replicon that is suitable for this purpose.
  • a truncated fusidic acid marker (comprised of the first 1/3 of the gene) would be ligated to the latter 2/3 of the cat gene and this construct inserted into pRN5101.
  • Transformants of the fusidic acid resistant strain would first be selected by growth in the presence of erythromycin. Recombinants which rescue the chloramphenicol marker into the plasmid result from homologous recombinations within the fusidic acid marker (thereby inactivating this gene) and cat gene sequences.
  • Recombination within the cat gene sequences can result in an active chloramphenicol marker residing in the host genome; this will not inactivate the fusidic acid marker. Both would be selected by growth in the presence of chloramphenicol. Growth of the recombinants at the non-permissive temperature in the absence of chloramphenicol would cure the strain of the plasmid, i.e. eliminate the plasmid form the bacterial strain. The cured strains would then be screened for sensitivity to fusidic acid to select for the appropriate recombinant.

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Abstract

L'activité de protéases résiduelles dans des souches AP-/NP- de Bacillus est due à deux protéases additionnelles, une protéase de sérine résiduelle (RSP) et une protéase de cystéine résiduelle dépendant de sulfhydryle (RCP) qui, ensemble, sont responsables de la dégradation de protéines recombinantes dans des cultures de B. subtilis (AN-/NP-). Le développement d'une analyse a permis l'identification et le développement de souches mutantes de B. subtilis qui sont déficientes non seulement en protéases neutres et alcalines mais également en l'une des deux protéases résiduelles ou les deux. Ces souches sont particulièrement bien appropriées pour être utilisées comme organismes hôtes pour la production de protéines hétérologues qui sont susceptibles à l'activité des protéases résiduelles.
EP19890906977 1988-05-05 1989-03-14 Protease-deficient gram-positive bacteria and their use as host organisms for the production of recombinant products Withdrawn EP0370103A4 (en)

Applications Claiming Priority (2)

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US19048388A 1988-05-05 1988-05-05
US190483 1988-05-05

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EP0370103A1 true EP0370103A1 (fr) 1990-05-30
EP0370103A4 EP0370103A4 (en) 1991-11-27

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JP (1) JPH03500606A (fr)
AU (1) AU622916B2 (fr)
DK (1) DK1590A (fr)
FI (1) FI900045A0 (fr)
HU (1) HUT53154A (fr)
IL (1) IL89767A0 (fr)
NZ (1) NZ228424A (fr)
PT (1) PT90463A (fr)
WO (1) WO1989010976A1 (fr)
ZA (1) ZA892325B (fr)

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US5143846A (en) * 1988-03-17 1992-09-01 The General Hospital Corporation Protease deficient bacterial hosts
US5294542A (en) * 1991-03-19 1994-03-15 Omnigene, Inc. Residual protease-III
IL102259A0 (en) * 1991-07-01 1993-01-14 Amgen Inc Isolation and characterization of a protease from streptomyces lividans
US5288931A (en) * 1991-12-06 1994-02-22 Genentech, Inc. Method for refolding insoluble, misfolded insulin-like growth factor-I into an active conformation
ATE275970T1 (de) 1993-10-05 2004-10-15 Celltech Pharmaceuticals Ltd Impfstoffzusammensetzungen
GB9324529D0 (en) * 1993-11-30 1994-01-19 Univ Singapore Biological control agents
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PT90463A (pt) 1989-11-30
AU3765189A (en) 1989-11-29
NZ228424A (en) 1992-06-25
WO1989010976A1 (fr) 1989-11-16
EP0370103A4 (en) 1991-11-27
HU894054D0 (en) 1990-07-28
ZA892325B (en) 1990-03-28
HUT53154A (en) 1990-09-28
FI900045A (fi) 1990-01-04
AU622916B2 (en) 1992-04-30
DK1590A (da) 1990-02-05
JPH03500606A (ja) 1991-02-14
IL89767A0 (en) 1989-09-28
FI900045A0 (fi) 1990-01-04
DK1590D0 (da) 1990-01-04

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