EP0148845A1 - Vecteur de clonage, son procede de preparation, ainsi qu'un procede pour concentrer et purifier des proteines de produits produites par le vecteur de clonage - Google Patents

Vecteur de clonage, son procede de preparation, ainsi qu'un procede pour concentrer et purifier des proteines de produits produites par le vecteur de clonage

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Publication number
EP0148845A1
EP0148845A1 EP84901674A EP84901674A EP0148845A1 EP 0148845 A1 EP0148845 A1 EP 0148845A1 EP 84901674 A EP84901674 A EP 84901674A EP 84901674 A EP84901674 A EP 84901674A EP 0148845 A1 EP0148845 A1 EP 0148845A1
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EP
European Patent Office
Prior art keywords
cell
transformant
product protein
plasmid
cells
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EP84901674A
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German (de)
English (en)
Inventor
Gordon L. Williams
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BIOVEC TECHNOLOGY Inc
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BIOVEC TECHNOLOGY Inc
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Publication of EP0148845A1 publication Critical patent/EP0148845A1/fr
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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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • This invention relates to stable cloning vec ⁇ tors involving inverted repeated sequences of DNA be ⁇ tween which is inserted an active origin of DNA repli ⁇ cation, to a method of constructing such cloning vec- tors, to the use of transformant cells to produce foreign product protein derived from the stable cloning vectors, and to a method of purifying and concentrating product proteins transported and bound to the cell surface which are produced by-the cloning vectors.
  • HLS host cell derived signal DNA sequences or hydrophobia leader sequences
  • HLS hydrophobia leader sequences
  • AGT transitional start signal
  • the signal sequences are clipped off enzymatically by selective cleavage of the signal sequence due to a specific signal peptidase enzyme coded by the bacterial host cell, resulting in release of the product protein from the surface of the cell.
  • a specific signal peptidase enzyme coded by the bacterial host cell resulting in release of the product protein from the surface of the cell.
  • Kroyer, Gray and Chang Genetic Cell Technology 1, 197- 205 (1982); Palva, et al., Proc. Nat. Acad. Sci. 79 (18), 5582-6 (1982). his requirement is fairly restrictive, as signal peptidase enzymes are highly specific in their activity.
  • bifunctional chimeric plasmid vectors for expressing foreign proteins is not novel. Constructs similar, but not identical, to pLF119 are described by Horinouchi and Weisblum, J. Bacteriol. 150 (2), 815-25, (1982). Their constructs, as in all other cloned constructs employed for this purpose, avoid the use of inverted repeated segments of DNA in form of palindromic DNA, or transposon structure. In B ⁇ subtilis, E. coli and other bacteria, the presence of inverted repeated DNA sequences has been recognized as a source of instability of the plasmid molecule. Hagan and Warren, Gene 19(1), 147-51 (1982); Hutchison, Sachter and Halvorson, Proc. Intl.
  • the present invention provides a means of constructing and utilizing sequences of DNA which lack homology with chromosomal DNA and which contain inverted repeated (palindromic) sequences of base coding, separated by an active replication origin site, to express foreign genes in bacterial or other cultured cells.
  • sequences of DNA may be derived from chimeric plasmids carrying the DNA for the product protein, the signal sequence and plasmid replicative genetic origin material known to be functional in the host cell or in the form of transposable DNA insertion sequences where active terminal repeated DNA sequences
  • This invention also relates to a method of purifying and concentrating product protein secreted through but bound to the cell wall, with subsequent release of the product protein by change of environmental conditions surrounding the cell.
  • a signal sequence from other than the host cell is used for mediating membrane transport of the product protein but retaining it bound to the cell wall.
  • the E_ j _ coli AMP signal sequence adjacent to the DNA coding for the desired product protein is used to mediate transport of the desired product protein in gram-positive host cells. In these cells, the product protein remains bound to the cell wall until released by change in environmental conditions surrounding the cells. ' '
  • membrane transport of the product protein occurs because of similarities between a 23 amino acid signal sequence (see Fig. 5) located at the start of the E ⁇ coli ⁇ -lactamase protein and certain polypeptide signal sequences found at the front of proteins which B ⁇ subtilis naturally secretes.
  • the 23 amino acid sequence on the E_ ⁇ coli ⁇ -lactamase is sufficiently similar to the signal sequences of B ⁇ subtilis to permit it to mediate transport of the protein across the membrane of the B. subtilis cell;
  • the E ⁇ colis signal sequence is not recognized by the enzyme responsible for releasing the protein at the surface of the cell. This results in the protein remaining bound to the cell surface where it accumulates in large quantities as ⁇ -lactamase is made and transported by the cell.
  • cloning vector pLF119 ATCC Deposit Accession No. 39380
  • similar vectors which contain inverted repeated DNA sequences to induce over-expression of product protein in gram-positive cells, particularly EL. subtilis and the use of gram-positive bacteria incorporating the cloning vector described to facilitate recovery and release of cell-bound product protein.
  • Fig. 1 schematically illustrates the method described in the application for preparation of the cloning vector in the form of a chimeric plasmid ⁇ pLF119);
  • Fig. 2 illustrates a map of the chimeric plasmid pLF119 determined by restriction enzyme analysis
  • Fig. 3 is a photomicrograph of B ⁇ subtilis strain BR151: Electron microscopy at 4500 X magnification;
  • Fig. 4 is a photomicrograph of B ⁇ subtilis strain BR151 expressing secreted E_ j _ coli ⁇ -lactamase product following introduction of pLF119 plasmid DNA: Electron microscopy at 4500 X magnification;
  • Fig. 5 is the nucleotide sequence of the pBR322 ⁇ -lactamase signal or hydrophobic leader sequence
  • Fig. 6 shows electrophoresis patterns for proteins recovered following release of E ⁇ coli ⁇ - lactamase from the cell surface.
  • O PI iAr O PI iAr .
  • WIPO Fig. 7 is a graph of amount vs. time for the production of released secreted ⁇ -lactamase gene pro ⁇ duct determined quantitatively by nitrocefin spectro- photometric assay.
  • the E_ j _ coli ⁇ -lactamase gene pro- duct was produced by B ⁇ subtilis BR151 carrying DNA introduced on plasmid pLF119 grown under various antibiotic pressures compared to control cells receiving similar treatment, including BR151 cells with no external DNA, BR151 cells carrying pC194 plasmid DNA, and BR151 cells carrying pWRlOl plasmid DNA.
  • the spectrophotometric assay was carried out according to the method described in Ross, Methods in Enzy ology (43), 69-85 (1975).
  • Fig. 8 is a photomicrograph of the "Q" form DNA derived from the plasmid pLF119 denoted as the "quas id” form of DNA derived from plasmid pLF119 in B. subtilis BR151: Electron microscopy at 225,000 X magnification;
  • Fig. 9 shows an electrophoresis pattern of the DNA band (shown by the arrow) of the "quasmid" illustrated in Fig. 8;
  • Fig. 10 describes generation of the "Q" form DNA from inverted DNA sequences flanking a replicative origin.
  • a cloning vector which is capable of replicating in host cells, such as E_ ; _ coli and B_j_ subtilis, and which contains inverted palindromic sequences of base coding separated by an active replication origin site capable of expressing foreign genes in the host cell.
  • the cloning vector may be (1) in the form of chimeric plasmid carrying the DNA for the foreign gene, a signal sequence and plasmid replicative genetic origin material known to be functional in the host cell so that the plasmid is capable of replicating in the host cell and expressing the foreign gene, or (2) in the form of a transposable DNA insertion sequence introduced on plasmid, viral or other DNA vectors.
  • the plasmid vector pBR322 is the most widely used and versatile of the plasmid cloning vectors and contains both ampicillin (AMP) and tetracycline (T ⁇ T) resistance genes and a number of restriction sites. Its complete nucleotide sequence is known. See Maniatis, T., et al.. Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, 1982. Also see Sutcliff, J. G., "pBR322 Restriction Map Derived from the DNA Sequence: Accurate DNA Size Markers up to 4361 Nucleotide Pairs Long," Nucleic Acid Research 5, 2721, 2728 (1978). The plasmid vector pC194 is also widely used and its complete nucleotide sequence known. Horimuchi and Weisblum, J., J. Bacteriol. 150 (2), 815-25 (1982).
  • Plasmid pLF119 was constructed, as illustrated in Fig. 1, by ligating a 1.7 Kb Clal fragment of Staph.
  • the structure of the plasmid was confirmed by restriction enzyme mapping. Characteristic Pstl and Hind III cleavage patterns, as well as Hae III, Ace I, and Hinf 1 single and double digests confirmed the structure of the plasmid molecule.
  • the plasmid is stable in E_j_ coli and transforms E ⁇ coli HB101 at a reduced efficiency relative to pBR322. The plasmid is efficiently retained under antibiotic pressure without structural alteration once transformation has been attained and can be amplified to high copy number levels in the E_ ⁇ coli host cells.
  • O FI O FI
  • Several techniques may be used to release the product protein from the cell surface in a controlled manner, including mild heating to about 50°-60°C, pH adjust- ment, or alteration of the ionic strength of the culture media.
  • the DNA sequence of interest for production of the foreign product protein may be cloned into the ⁇ -lactamase gene of pBR322 adjacent to the signal sequence end illustrated by Fig. 5.
  • ⁇ -lactamase itself was used as a specific example of a working system; however, other desired proteins or polypeptides may be produced in the same manner by introducing the appropriate DNA sequence into the ⁇ -lactamase sequence by linkers or unique cloning sites downstream from the signal sequence region of the gene.
  • To clone in another gene for the ⁇ -lactamase gene which is to be expressed in a secretory mode requires that splicing be done so that ⁇ -lactamase signal sequence be adjacent the structural gene of interest in the DNA sequence.
  • ⁇ -lactamase is produced at constituitive levels in the absence of antibiotic pressure and that ampicillin pressure amplifies expression of the foreign gene product.
  • the foreign gene in this instance ⁇ - lactamase, is secreted by the B ⁇ subtilis cells, remaining bound at the surface of the cells due to the absence of a proper signal peptidase to cleave the pBR322-derived signal sequence.
  • This accumulation of secreted, bound foreign product protein at the cell surface produces the "clumpy" phenotype illustrated in Fig. 4 in comparison to B_ j _ subtilis strain BR151 not carrying DNA derived from plasmid pLF119 as illustrated in Fig. 3.
  • the "clumpy" cells illustrated in Fig. 4 show the presence of ⁇ -lactamase enzyme activity as demon ⁇ strated by nitrocefin disc assay.
  • the culture supernatant broth did not exhibit ⁇ -lactamase activity until exposure of the cells illustrated in Fig. 4 in broth to 50°C for 10 minutes or to pH change.
  • ⁇ - lactamase activity then appeared in the supernatant fluid in quantities approaching 5 mg/ml of active pro ⁇ tein enzyme, as illustrated by Fig. 7.
  • Increased levels of ⁇ -lactamase were observed in the presence of ampicillin; however, significant quantities of the enzyme were present in the absence of antibiotic pressure.
  • Lanes 4 and 5 O PI gel analysis of proteins (Fig. 6, Lanes 4 and 5).
  • Lanes 1 and 5 are molecular weight standards.
  • Lane 6 is the SDS gel analysis of proteins in the supernatant from E ⁇ coli cells carrying pBR322 plasmid DNA.
  • Lane 3 is the same for B ⁇ subtilis BR151 cells carrying pC194 plasmid DNA.
  • a finding that the ⁇ -lactamase enzyme disappears on DEA ⁇ cellulose and is not released from the column confirmed the presence of the ⁇ -lactamase signal sequence. This would be expected of a molecule retaining strong hydrophobic regions.
  • the electron microscope confirmation of this structure is shown in Fig. 8.
  • the quasmid (1) replicates, (2) expresses genes, (3) is stably maintained in membrane complex in the bacterial cell, and (4) constitutes a novel method of maintaining and expressing foreign DNA in B ⁇ subtilis.
  • Lysis of BR151 B ⁇ subtilis cells carrying plasmid pLF119 produced no characteristic plasmid band following cesium chloride density gradient centrifugation. Probes of purified chromosomal DNA with CAT and AMP gene fragments from pC194 and pBR322 produced no evidence of chromosomal integration. Ampicillin and chloramphenicol resistance were not lost during curing with acridine orange in cells transformed with pLF119 DNA. The clumpy phenotype illustrated in Fig. 4 was produced in response to ampicillin pressure irrespective of the presence or absence of chloramphenicol.
  • Transformation of BR151 with chromosomal DNA from BR151 expressing AMP, CAM, and the clumpy phenotype illustrated by Fig. 4 did not result in a transfer of any of these traits to the progeny. Plasmid curing experiments did not result in loss of the traits, nor did any plasmid obtained by standard techniques of isolation from the cells.
  • Lane 1 is the molecular weight standard.
  • Lane 2 shows the electrophoresis pattern of the DNA bands of B ⁇ subtilis BR151 cells carrying pLF119 plasmid DNA-ampicillin amplified.
  • Lane 3 shows the same for B ⁇ subtilis BR151 cells carrying pC119 plasmid DNA without ampicillin amplification. Both S ] _ and single-stranded specific endonucleases degraded the bands; however, the bands were- resistant to many restriction endonucleases. Nicking the bands with DNAse did not alter the banding
  • Both the 1.1 Kb and the 10.0 Kb bands are produced by the host cell, whether by autonomous replication or degradation of other identified forms.
  • the 10.0 Kb band appears to represent the whole double- stranded plasmid pLF119. Alternatively, it could be linear double-stranded concatameric DNA produced in some fashion by the 1.1 Kb band.
  • the 10.0 Kb band is greatly enriched under AMP pressure, suggesting it is the source of the amplification of ⁇ -lactamase expression through increased copy number.
  • the 1.1 Kb band remains unchanged quantitatively during ampicillin amplification, it apparently is important in the development of the amplified secreting system. Cells which lack a 1.1 Kb band are unable to undergo f amplification of the 10 Kb band or increase expression of the gene product.
  • the cloning vector in the form of a chimeric plasmid, or transposable insertion sequence is introduced into the host cell by the known processes of genetic transformation, transfection, or . viral infection of the cells or protoplasts of the cell in a manner such that production and secretion to the cell surface of the product protein coded by the vector DNA ensues.
  • the host cells carrying- the cloning vector are isolated and cultured by conventional fermentation processes and produce a characteristic clumpy appearance when grown in liquid media, as illustrated by Fig. 4.
  • the "clumped" cells are the result of accumulation of product protein synthesized in the transformed host cells and secreted to the exterior of the cell where it remains bound in large quantities to the surface of the cell. Alteration of the surrounding environment of the host cell by pH, temperature change, or ionic strength releases the entire uncleaved product protein and signal sequence from the cell surface, leaving the transformed cell available to produce an additional crop of protein product.
  • the cells may be periodically harvested and returned to grow and produce additional product in a continuous culture mode.
  • the product protein released into the fluid medium may contain the signal sequence whose removal may require subsequent processing. Released product protein in the medium is present in active form at high concentration (see Fig. 6).
  • Levels of product protein production in the amplified system calculated by quantitative assay of ⁇ -lactamase enzyme released from log growth cells, ranged consistently between 2 million and 20 million active enzyme molecules produced per cell over 4-6 hours of active cell growth—substantially more product in a more pure form than can be produced in E ⁇ coli.
  • the transformant cells may be immobilized on a support surface for culture and production of the product protein as described, for example, in Mosbach, et al.. Nature, 302 (7) 543-45 (1983).
  • the bacterial hosts used in the examples included strains of E coli HB101 (ATCC No. 33694) described by Boyer et al., J. Mol. Biol. 41, 459-472 (1969), and B ⁇ Subtilis BR151 (ATCC No. 37705).
  • the buffers and media used are commercially available and were purchased from Difco Corporation.
  • DNA was isolated from pBR322 amplified in E. coli for 18 hours with chloram ⁇ phenicol. This DNA was further purified on a cesium chloride gradient. Analysis of the product on agarose and acrylamide gels indicated that it was plasmid DNA. Restriction fragments with AVA II matched those of pBR322. Restriction with Clal produced a single band of linear DNA representing the entire pBR322 sequence.
  • DNA from pC194 was isolated from B. subtilis and purified on cesium chloride. Restriction with Clal produced two bands of molecular weight, 1.7 Kb and 1.3 Kb, respectively. The 1.7 Kb fragment was cut from agarose gels and eluted by the "squeeze freeze" technique.
  • the 1.7 Kb fragment of pC194 was ligated with the linear pBR322 fragments by standard procedures, except that the alkaline phos ⁇ phatase treatment was omitted in order to obtain the- inverted pBR322 dimer unit.
  • the construct was trans ⁇ formed into E. coli HB101 and grown on LB media with 25 ⁇ g/ml CAM. Transformants appeared after two days, and 32 clones were initially screened from single col ⁇ ony isolates. The recombinants were selected for abil ⁇ ity to grow in 25 ⁇ g/ml ampicillin and inability to grow in 25 ⁇ g/ml tetracycline. Restriction enzyme map- ping was carried out by conventional procedures to con ⁇ firm the structure of the plasmid illustrated in Fig. 2. The restriction endonuclease used included PST I, Hinds III, Ace I and Hinf I.
  • B. subtilis strain BR151 was grown in HS media for 4-1/2 hours and transferred into LS media at 1:5 dilution. Cells were grown in LS media for 90 min ⁇ utes. 0.2 ⁇ g of DNA of pLF119 extracted from E. coli, HB101, were added to 1 mL of competent cells and incu ⁇ bated for 30 minutes at 37°C. The reaction was termi- nated by adding 2.5 ⁇ g of DNase dissolved in 0.15 M sodium chloride. Cells were incubated for 2 hours .at 37°C. for expression or drug resistance. Clones were plated on CAM/AMP fortified. TBAB plates for selection.
  • clones were selected from competent cell trans- formation and streaked on CAM/AMP TBAB plates. These clones were grown in 10 ml . broth culture containing 25 ⁇ g/ml CAM/AMP. The clone appearing to have the "clumpiest" growth chracteristic was chosen for ⁇ -lac ⁇ tamase assay. A culture of the chosen transfor ant was grown 2 hours in 10 ml PA plus 25 ⁇ g/ml CAM/AMP. Cells were pelleted and resuspended in l/20th volume of STE buffer. Part of the resuspended cells were heat treated at 55°C. for 15 minutes while the other half were left at room temperature.
  • pLF119 derived from E. coli HB101
  • B. subtilis strain BR 151 was transformed as previously described into B. subtilis strain BR 151.
  • the transformants were screened for acquisition of resistance to 25 ⁇ g/ml AMP and CAM. Clones which satisfied the antibiotic resistance cri- teria were grown in PA broth under ampicillin pressure and visually screened for production of pronounced "clumpy" phenotype as shown in Fig. 4.
  • Cultures of B. subtilis strain . BR151 carrying the introduced pLF119 plasmid which had been grown on CAM/AMP containing TBAB plates were streaked onto TBAB plates containing CAM/AMP, CAM, AMP, and a plate with no antibiotics and the plates incubated overnight at 37°C.
  • Cells were removed from each plate and used to inoculate 40 ml of a PA broth containing CAM/AMP, CAM, AMP, and no anti- biotics. Again, cells were taken from each plate and centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the cells resuspended in 5 ml STE buffer and incubated at 50°C. for 15 minutes. The cells were pelleted and the supernatant saved to test for ⁇ -lactamase activity. The pellet was redissolved in 500 ⁇ g STE buffer and 500 ⁇ g/ml lysozome and incu ⁇ bated for 30 minutes at 37°C.
  • the DNA was collected by cen- trifugation at 10K rpm for 20 minutes, resuspended in 2 ⁇ l ammonium acetate, and reprecipitated with three volumes of ethyl alcohol.
  • the DNA pellet was redissolved in 100 ⁇ l TE buffer and stored at -20°C.
  • Protein extracts from each culture were assayed for ⁇ - lactamase activity.
  • One hundred ⁇ l of each sample was mixed with 900 ⁇ l nitrocefin solution in 0.1 molar ammonium acetate and read at various times by spectro- phometric assay techniques.
  • Controls of B. subtilis strain BR151, containing plasmid pC194 and BR151 containing plasmid pWRlOl were also run with a ⁇ - lactamase standard. The results are as shown in the graph of Fig. 7.

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Abstract

Nouveau vecteur de clonage caractérisé par des séquences d'ADN inversées répétées entre lesquelles est insérée une origine de duplication active. L'utilisation dudit vecteur de clonage, lorsqu'il est introduit dans une cellule hôte, produit une protéine de produit étrangère dont la séquence signal est dérivée d'un point de départ autre que la cellule hôte, la séquence signal servant de médiateur pour le transport membranaire de la protéine de produit mais retenant celle-ci liée à la surface cellulaire jusqu'à libération par une modification des conditions ambiantes dans lesquelles se trouvent les cellules hôtes. Ce système offre un moyen de régulation de la libération de protéines de produit en vue d'une concentration sous une forme très purifiée.
EP84901674A 1983-06-29 1984-03-30 Vecteur de clonage, son procede de preparation, ainsi qu'un procede pour concentrer et purifier des proteines de produits produites par le vecteur de clonage Withdrawn EP0148845A1 (fr)

Applications Claiming Priority (2)

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US50839183A 1983-06-29 1983-06-29
US508391 1983-06-29

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EP0148845A1 true EP0148845A1 (fr) 1985-07-24

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EP84901674A Withdrawn EP0148845A1 (fr) 1983-06-29 1984-03-30 Vecteur de clonage, son procede de preparation, ainsi qu'un procede pour concentrer et purifier des proteines de produits produites par le vecteur de clonage

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EP (1) EP0148845A1 (fr)
JP (1) JPS60501688A (fr)
AU (1) AU2860384A (fr)
IT (1) IT1178489B (fr)
WO (1) WO1985000185A1 (fr)

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IT1177378B (it) * 1984-12-11 1987-08-26 Anic Spa Vettore plasmidico psm112 ad espressione in bacillus subtilis

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See references of WO8500185A1 *

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JPS60501688A (ja) 1985-10-11
WO1985000185A1 (fr) 1985-01-17
AU2860384A (en) 1985-01-25
IT8421612A0 (it) 1984-06-26

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