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  • 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
  • C12N15/67—General methods for enhancing the expression
  • C12N15/69—Increasing the copy number of the vector
• • 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

Definitions

• the invention relates to an origin of plasmid replication, and to plasmids comprising said origin.
• Plasmids are fragments of extrachromosomal circular DNA, transferable from one bacterium to another, and whose replication takes place independently of that of the bacterial chromosome.
• a given plasmid can be present in a large number of copies inside a bacterial cell.
• the copy number is a genetic characteristic of each plasmid.
• ColE1 type plasmids such as plasmids of the pBR, pUC families, etc.
• the number of copies is under control of a region of DNA corresponding to the origin of replication of the plasmid (ORI) which extends approximately between bases 2940 and 3130 (numbering of the bases of PBR322 proposed by PEDEN [Gene, 22 (1983) 277-280].
• ORI origin of replication of the plasmid
• a part of this region, located between bases 2970 and 3089 is transcribed in RNAs called ARNI and RNAII, RNAI, in particular, would play a role in regulating the copy number of
• Plasmids are commonly used in genetic engineering as vectors for the cloning and expression of foreign genes in bacteria.
• a conventional technique for obtaining a large quantity of plasmids is to add chloramphenicol to bacterial cultures, which prevents the cell multiplication by blocking translation, without affecting plasmid replication.
• chloramphenicol also interferes with the translation of the foreign gene, this method is not optimal when an increase in the expression of said gene is sought.
• Plasmids carrying a mutation affecting the number of copies have been described in the literature.
• BOROS et al. [Gene, 30, 257-260 (1984)] thus describe a mutant plasmid derived from pBR322.
• the number of copies of this plasmid per cell is increased by approximately 200 times compared to the number of copies of pBR322. This increase in the number of copies results from a G ⁇ T transversal located at position 3075 on the HinfI fragment 2846-3363, near the 2 'end of the sequence transcribed into RNA I.
• the inventors have sequenced the origins of replication of the mutated plasmids, and have located the corresponding mutations.
• the subject of the present invention is a DNA sequence, derived by mutation from an origin of replication of the ColE1 type, which sequence is characterized in that it comprises at least one of the following mutations, defined with respect to wild type ColE1 origin:
• said sequence carries the two mutations defined above.
• FIG. 1 shows in (la) the sequence that region 2970-3089 of an origin of wild replication of pBR 322, and in (lb) a sequence in accordance with the invention, carrying the two mutations defined above.
• a mutated sequence in accordance with the invention could be inserted into other plasmids in place of the corresponding sequence of the origin of initial replication, and that this resulted in an increase in the number copies of the recombinant plasmids thus obtained.
• the present invention therefore includes plasmids characterized in that their origin of replication, ColE1 type, includes a mutated sequence as defined above.
• Figures 2, 2 and 4 show plasmids according to the invention respectively called pLMl, pLM2 and pLM3.
• the plasmids pLM1, pLM2 and pLM3 derive from the plasmid pUC9 by mutation, as defined above, from the origin of replication, and also contain the complete gene for ⁇ -galactosidase, framed by a polylinker, and placed under the control of the promoter of the LacZ gene.
• plasmids in accordance with the invention can be easily obtained, for example, from plasmids carrying an origin of replication of the ColE1 type, by a directed mutagenesis technique, indeed from a plasmid such as pML1 by excising from the latter the mutated DNA segment and inserting it in place of the corresponding wild-type DNA segment of the recipient plasmid. It will appear to a person skilled in the art that it is also possible to obtain a DNA segment in accordance with the invention by oligonucleotide synthesis, according to techniques known in themselves and to insert it into the recipient plasmid.
• FIG. 5b represents the plasmid pARA 15 thus obtained from a plasmid called pARA 14
• FIG. 5a which is an expression vector comprising the origin of replication of pBR322, a gene for resistance to ampicilin and a promoter inducible by arabinose.
• Receptor plasmids which can be used for the construction of plasmids conforming to the invention are generally, all the plasmids compatible with an origin of replication of the ColE1 type
• the number of copies of the plasmids thus obtained is at least multiplied by 10 with respect to the corresponding "wild" plasmids.
• the expression of foreign genes placed in these plasmids increases in the same proportions.
• the promoters that control the expression of these genes remain inducible.
• the host strains suitable for the multiplication of the plasmids in accordance with the invention and for the expression of the genes carried by these plasmids are the same as those allowing the multiplication of the corresponding wild plasmids and the expression of the genes which they carry, and the behavior and growth of the strains transformed by the plasmids in accordance with the invention are identical to those of the strains carrying the wild plasmids.
• the present invention further relates to a process for the multiplication of plasmids in accordance with the invention, which process is characterized in that, in a first step, the transformation of an appropriate host bacterial strain is carried out by at least one of said plasmids, and in a second step, the culture of said bacterial strain is carried out.
• the invention also includes:
• a method of amplifying a DNA sequence which method is characterized in that, in a first step, said sequence is inserted into a plasmid according to the invention, and in that, in a second step, one proceeds to the multiplication of said plasmid, as indicated above.
• a process for the production of polypeptides by genetic engineering which process is characterized in that, in a first step, the gene encoding said polypeptide is inserted into a plasmid according to the invention, in a second step, one proceeds to the transformation of an appropriate bacterial host strain with said plasmid, and in a third step, one proceeds to the culture of said bacterial strain under conditions suitable for the expression of said gene.
• the plasmid pARA 15 is derived from the plasmid pARA 14 by replacing the origin of initial replication of the latter (which is that of pBR322) by the origin of mutated replication in accordance with the invention.
• a 2060 bp SstI / BglI fragment of pARA 14, comprising its origin of replication is excised and replaced by a 1430 bp SstI / BglI fragment from pLM3, comprising the origin of replication in accordance with the invention.
• FIG. 5 represents the plasmid pARA 14 (5a), carrying an origin of wild-type replication, and the plasmid pARA 15 (5b) carrying an origin of replication in accordance with the invention.
• the foreign genes which it is desired to express are under the control of a promoter inducible by arabinose.
• the DNA was extracted from cultures of E. coli TG1 cells (2 ml), harvested at 2.3 U OD 6 0 0 .
• the cells were lysed with a mixture of lysozyme, detergent and sodium hydroxide. After centrifugation, the plasmid DNA contained in the supernatant was separated from the proteins by phenol extraction followed by treatment with chloroform.
• the pure plasmid DNA is then precipitated with ethanol, the precipitate is centrifuged and dried under vacuum, then redissolved in 50 ⁇ l of buffer (Tris 10 mM, EDTA ImM pH8) for each sample. Extracts of E. coli TG1 cells containing either the plasmid pARA 14 (control strains) or the plasmid pARA 15 were prepared in this way.
• the ⁇ -lactamase (expressed from the bla gene present on each of the plasmids pARA 14 and pARA 15 and conferring resistance to ampiciiline) produced in the exponential growth phase was assayed for an E. coli strain TG1 containing either the plasmid pARA14 or the plasmid pARA15.
• control plasmids are on the one hand pUC 9 and on the other hand the plasmid pARA14PR107 ⁇ -gal which differs from pARAl5PR107 ⁇ -gal only by its origin of replication, which is that of the "wild type", from pBR322).
• ⁇ -galactosidase assay protocol 5 ml of LB medium supplemented with 100 ⁇ g of ampiciilin per ml, were inoculated with 250 ⁇ l of a saturated culture and then incubated with shaking at 37 oC. When the DO 600 reached 3.4, the induction is carried out with 2 mM of IPTG for the strains hosting pCU9 or pLM3, eu 0.2% of arabinosis for the strains hosting pARA14PR107 ⁇ -gal or pARA15PR107 ⁇ -gal. The ⁇ -gaiactosida ⁇ e activity is then assayed in culture samples taken at different incubation times (0 min, 40 min, 120 min.)
• the level of ⁇ -galactosidase obtained in the cell pellets is approximately 16 to 20 times higher with pLM3 than with pUC9, and approximately 2.5 times higher with pARA 15 that with pARA 14.

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• 1991
  • 1991-07-12 FR FR9108836A patent/FR2678947B1/fr not_active Expired - Fee Related
• 1992
  • 1992-07-10 US US08/175,354 patent/US5565333A/en not_active Expired - Fee Related
  • 1992-07-10 EP EP92916263A patent/EP0594776A1/de not_active Withdrawn
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