Host cell for producing proteins
View Patent ↗Disclosed is a host cell for producing a recombinant peptide, polypeptide or protein of interest, wherein the host cell includes at least 2 copies of a nucleic acid sequence encoding a poison protein; and to the use thereof for producing peptides, polypeptides or proteins of interest.
1. Bacterial host cell comprising at least 2 chromosomal copies of a nucleic acid sequence encoding a poison protein, provided that said at least 2 copies are in a different replicon than a nucleic acid sequence encoding the antidote protein to the poison protein, wherein said cell further comprises at least one copy of a nucleic acid sequence encoding the antidote protein to the poison protein, and wherein said nucleic acid sequence encoding the antidote protein is carried by a plasmid further comprising an expression system wherein a nucleic acid sequence encoding a recombinant peptide, polypeptide or protein of interest is or may be inserted.
2. Host cell according to claim 1 , wherein said poison protein is CcdB, encoded by SEQ ID NO: 1 or any nucleic acid sequence having at least 75% identity with SEQ ID NO: 1.
3. Host cell according to claim 1 , wherein said antidote protein is CcdA, encoded by SEQ ID NO: 13 or any nucleic acid sequence having at least 75% identity with SEQ ID NO: 13.
4. Host cell according to claim 1 , wherein said expression system comprises a promoter selected from the group comprising a T7 promoter, Ptrc, Para and Plac.
5. Host cell according to claim 1 , wherein said expression system comprises a T7 promoter and the host cell further comprises a T7 RNA polymerase gene.
6. Host cell according to claim 1 , wherein said expression system comprises a T7 promoter and the host cell further comprises a genetically modified phage inserted within its genome.
7. Host cell according to claim 1 , wherein said expression system comprises a T7 promoter and the host cell further comprises a genetically modified phage defined as a phage wherein:
a T7 expression system is inserted,
the S, R, and/or the Q genes are inactivated, and
the Int and/or Xis gene are inactivated.
8. Host cell according to claim 1 , further comprising inactivation of at least one of the genes conA, galK, araB, araA, lon, ompT, rcsA, hsdR, mrr, endA and recA.
9. Host cell according to claim 1 , further comprising at least two copies of a nucleic acid sequence encoding a functional GyrA protein.
10. A kit comprising:
a host cell comprising at least 2 chromosomal copies of a nucleic acid sequence encoding a poison protein, provided that said at least 2 copies are in a different replicon than a nucleic acid sequence encoding the antidote protein to the poison protein, and
a vector comprising at least one copy of the nucleic acid sequence encoding the antidote protein and at least one copy of an expression system wherein a nucleic acid sequence encoding a recombinant peptide, polypeptide or protein of interest is or may be inserted.
11. The kit according to claim 10 , wherein the nucleic acid sequence encoding a peptide, polypeptide or protein of interest is under the control of a promoter selected from the group comprising a T7 promoter, Ptrc, Para and Plac.
12. A method for producing a recombinant peptide, polypeptide or protein of interest, wherein said method comprises:
cultivating the host cell of claim 1 , wherein the host cell further comprises at least one copy of the nucleic acid sequence encoding the peptide, polypeptide or protein of interest,
and recovering the peptide, polypeptide or protein of interest.
13. The method according to claim 12 , wherein the recombinant protein is a secreted protein, a transmembrane protein or a protein which is toxic for the bacterial strain.
14. The host cell of claim 5 , wherein the T7 RNA polymerase gene is inserted in a genome of the host cell.
15. The host cell of claim 5 , wherein the T7 RNA polymerase gene is inserted in a genome of the host cell in a T7 expression system.