Mgmt-based method for obtaining high yeilds of recombinant protein expression

Patent metadata

Field	Value
Publication number	US-2016017367-A1
Application number	US-201514811079-A
Country	US
Kind code	A1
Filing date	Jul 28, 2015
Priority date	Dec 9, 2010
Publication date	Jan 21, 2016
Grant date	—

Abstract

The present invention relates to a novel enhancer of protein production in host cells. It discloses a vector for expressing recombinant proteins in these cells, comprising a nucleotide sequence encoding a) a secretion peptidic signal, b) a 6-methylguanine-DNA-methyltransferase enzyme (MGMT, EC 2.1.1.63), a mutant or a catalytic domain thereof, and c) a recombinant protein. Said MGMT enzyme is preferably the so-called SNAP protein.

First claim

1 . A vector for expressing recombinant proteins in host cells, comprising a nucleotide sequence encoding in a single open reading frame, from 5′ to 3′:
a) a peptidic secretion signal which is functional in said host cells,
b) a 6-methylguanine-DNA-methyltransferase enzyme (MGMT), a catalytic domain, or a mutant thereof, and
c) a recombinant protein.

2 . The expression vector according to claim 1 , wherein said MGMT enzyme or MGMT mutant is the protein of SEQ ID NO:4, or a homologous sequence thereof that is at least 80% identical to SEQ ID NO:4.

3 . (canceled)

4 . The expression vector according to claim 1 , wherein said open reading frame is operatively associated with an inducible promoter which is functional in the same host cell as the peptidic signal is.

5 . The expression vector according to claim 4 , wherein said secretion peptidic signal and said inducible promoter are functional in non-vertebrate cells.

6 . The expression vector according to claim 4 , wherein said secretion peptidic signal and said inducible promoter are functional in vertebrate cells.

7 . (canceled)

8 . The expression vector according to claim 1 , wherein the recombinant protein is selected from the group consisting of:
bacterial or viral immunogenic proteins selected from the EDIII protein from Dengue, Japanese encephalitis (JE), Tick-borne encephalitis (TBE), Yellow fever (YF), Usutu (USU), Rocio, Murray Encephalitis (MVE), Wesselbron (WSL), Zika and West Nile (WN) viruses, the nucleoprotein N from Rift Valley Fever (RVF) and Toscana (TOS) viruses, the soluble form of the E2 envelope protein from the Chikungunya virus, and the soluble form of the E envelope protein of the West-Nile virus blood factors, anticoagulants, growth factors, hormones, therapeutic enzymes and monoclonal antibodies and cytokines, anti-tumoral proteins, microbial, viral and/or parasite polypeptides, and antigens.

9 . The expression vector according to claim 1 , wherein said recombinant protein is selected from the group consisting of IFNα, Granzyme M, FasL, SSX2, NERCMSL, hSULF2 ΔTMD and CNTN4.

10 . The expression vector according to claim 1 , wherein said MGMT enzyme is encoded by the DNA sequence of SEQ ID NO:3, or SEQ ID NO:68.

11 . (canceled)

12 . The expression vector according to claim 1 , further encoding at least one peptidic cleavage site, which is preferably located between the MGMT enzyme, or mutant or catalytic domain thereof, and the recombinant protein.

13 - 14 . (canceled)

15 . The vector according to claim 1 , encoding in frame from 5′ to 3′:
a peptidic BiP insect signal or a BiP-like peptide signal,
a MGMT protein of SEQ ID NO: 4,
at least one enterokinase cleavage site or proTEV peptidic cleavage site,
a poly-Histidine label, and,
two spacer sequences having the amino acid sequence Glycine-Glycine-Glycine-Serine (GGGS).

16 - 23 . (canceled)

24 . A fusion polypeptide encoded by the vector of claim 1 .

25 - 26 . (canceled)

27 . The fusion polypeptide according to claim 24 , further comprising a label located at the C terminal end of the recombinant protein.

28 . A non-vertebrate recombinant cell which is stably transfected by the expression vector of claim 1 .

29 . The stably transfected cell according to claim 28 , wherein it is an insect cell.

30 . The stably transfected cell according to claim 28 , wherein it is a Drosophila melanogaster cell.

31 . (canceled)

32 . A vertebrate recombinant cell which is stably transfected by the expression vector of claim 1 .

33 . The stably transfected cell according to claim 32 , wherein it is a mammalian cell.

34 . (canceled)

35 . A method of enhancing expression of recombinant protein(s) comprising expressing the vector of claim 1 in non-vertebrate cells.

36 . The method according to claim 35 , wherein said MGMT enzyme or MGMT mutant enzyme is the protein of SEQ ID NO:4, or a homologous sequence thereof that is at least 80% identical to SEQ ID NO:4.

37 . (canceled)

38 . A method to produce recombinant protein in cell culture, comprising the steps of:
a) providing the expression vector of claim 1 , b) introducing said expression vector into host cells, c) allowing for the expression of the nucleotide introduced in said host cells to produce the recombinant protein.

39 - 44 . (canceled)

45 . The expression vector according to claim 5 , wherein said secretion peptidic signal is a BiP insect signal or a BiP-like peptide.

46 . The expression vector according to claim 15 , further encoding a recombinant protein comprised between the MGMT protein and the cleavage site, said protein being preferably selected from the group consisting of IFNα, Granzyme M, FasL, SSX2, NERCMSL, hSULF2 ΔTMD and CNTN4.

Assignees

• Pasteur Institut

Inventors

• Despres Philippe
• Paulous Sylvie
• Crublet Elodie

Classifications

• C07K2319/21

  containing a His-tag · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C12N2800/107

  for mammalian · CPC title

• C07K2319/055

  containing a signal for localisation to secretory granules (for exocytosis) · CPC title