Method and system for the production of recombinant proteins by cells

Patent metadata

Field	Value
Publication number	US-10696997-B2
Application number	US-201515323891-A
Country	US
Kind code	B2
Filing date	Jul 6, 2015
Priority date	Jul 4, 2014
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

Abstract

A cassette sequence for the transformation of a host cell includes at least: a first nucleotide sequence encoding a peptide or protein of interest to be produced by the host cell. The first sequence is linked to a second nucleotide sequence providing resistance to a toxin or encoding an antitoxin peptide to the toxin. The nucleotide sequences are organized in such a way that production of the peptide encoded by the second nucleotide sequence(s) is translationally coupled to production of the peptide encoded by the first nucleotide sequence.

First claim

The invention claimed is:

1. A host cell comprising in its genome,
(i) a cassette sequence or a vector comprising said cassette sequence, wherein the cassette sequence or vector comprising said cassette sequence is expressed in said host cell, the cassette sequence comprising:
a first nucleotide sequence encoding a peptide of interest,
a second nucleotide sequence encoding an antitoxin peptide to a toxin, wherein the antitoxin peptide is only encoded within the host cell by this second nucleotide sequence, and
a third nucleotide sequence encoding a linker peptide having a length comprising between 2 and 500 amino acids;
wherein the first, second, and third nucleotide sequences are organized to encode a fusion protein comprising, from N- to C-terminal, the protein of interest, the linker peptide, and the antitoxin; and
(ii) one or more copies of a fourth nucleotide sequence encoding a toxin which is toxic to the host cell, wherein said fourth nucleotide sequence is expressed in said host cell.

2. The host cell of claim 1 , wherein the linker peptide comprises a sequence cleavable by a protease.

3. The host cell of claim 2 , wherein the protease is the TEV protease.

4. The host cell of claim 1 , wherein the linker peptide is an auto-cleavable peptide.

5. The host cell of claim 1 , wherein the toxin is an herbicide or fungicide.

6. The host cell of claim 1 , wherein the toxin/antitoxin peptides that are expressed in said host cell are selected from the group consisting of CcdB/CcdA, Kid/Kis (PemK/PemI), ParE/ParD, MazE/MazF, RelE/RelB, YafO/YafN, HipA/HipB, Doc/PhD, VapCNapB, VapDNapX, HicA/HicB, YoeB/YefN, YafQ/DinJ, Tse2/Tsi2(PA2702/PA2703), TseI(PA1844)/TsiI, Tse3(PA3484)/Tsi3, C-terminal portions of Rhs (Rhs-CT) or CdiA (Cdi-CT) peptides/associated immune peptides RhsI or CdiI, or bacteriocins peptides.

7. The host cell of claim 6 , wherein the toxin/antitoxin peptides are Kid/Kis peptides.

8. The host cell of claim 1 , wherein the toxin is lethal for the host cell.

9. The host cell of claim 1 , wherein the vector is a plasmid.

10. The host cell of claim 1 , wherein the host cell is a bacterial cell.

11. The host cell of claim 10 , wherein the bacterial cell is E. coli.

12. The host cell of claim 1 , wherein the host cells is a eukaryotic cell.

13. A method for improving the expression of a peptide of interest, said method comprising:
culturing the host cell of claim 1 in culture medium under conditions in which the fusion protein comprising the peptide of interest is expressed.

14. The method of claim 13 , said method further comprising recovering the fusion protein comprising the peptide of interest or the peptide of interest from the host cell or the culture medium.

Assignees

• Univ Bruxelles

Inventors

• Van Melderen Laurence
• Dumont Baptiste J S
• Hallaert Thibaut
• Vanwalleghem Gilles
• Goffin Philippe

Classifications

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12N15/65

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2319/92

  containing an intein ("protein splicing")domain · CPC title