Mutant E. coli strains, and their use for producing recombinant polypeptides
AssigneeCentre National De La Recherche Scientifique
InventorsMarc Dreyfus, Pascal Lopez
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Abstract
The invention concerns the use of Escherichia coli (E. coli) strains whereof the gene coding for the Rnase E comprises a mutation such that the enzyme produced when said mutated gene is expressed no longer has a degrading activity on mRNA, said mutation more significantly not affecting the growth of E. coli strains, for implementing a method for producing specific exogenous recombinant polypeptides.
Claims11
A process for producing predetermined recombinant polypeptides or proteins, comprising expressing said polypeptides or proteins in Escherichia coli (E. coli) strains whose gene encoding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene no longer possesses activity for degrading messenger RNA (m-RNA), said mutation not significantly affecting growth of the said E. coli strains, and wherein said mutation corresponds to the substitution of the guanine G at position 2196 of SEQ ID NO:1, by a thymidine T, so as to create a stop codon TAA situated at the positions 2196 to 2198 of SEQ ID NO:
- 1.
E. coli strains transformed such that they contain an inducible expression system, and whose gene encoding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene no longer possesses activity for degradation of m-RNA, this mutation not significantly affecting growth of the said E. coli strains, and wherein said mutation corresponds to the substitution of the guanine G at position 2196 of SEQ ID NO:1, by a thymidine T, so as to create a stop codon TAA situated at the positions 2196 to 2198 of SEQ ID NO:
- 1.
A process for producing predetermined recombinant polypeptides, characterized in that it comprises:
- transforming E. coli strains with a plasmid vector containing a nucleotide sequence encoding one or several recombinant polypeptides, wherein said E. coli strains are transformed such that they contain an inducible expression system, and whose gene encoding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene no longer possesses activity for degradation of m-RNA, said mutation not significantly affecting growth of said E. coli strains, said mutation corresponds to the substitution of the guanine G at position 2196 of SEQ ID NO:1, by a thymidine T, so as to create a stop codon TAA situated at the positions 2196 to 2198 of SEQ ID NO:
- 1,
- culturing the transformed E. coli strains obtained in the preceding step, for a time sufficient to permit expression of the recombinant polypeptide or polypeptides in the E. coli cells, and
- recovery of the recombinant polypeptide or polypeptides produced during the preceding step.
Dependencies are inferred from the claim text.