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

Patent metadata

Field	Value
Publication number	US-10017769-B2
Application number	US-201615377554-A
Country	US
Kind code	B2
Filing date	Dec 13, 2016
Priority date	Dec 9, 2010
Publication date	Jul 10, 2018
Grant date	Jul 10, 2018

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

The invention claimed is:

1. An immunoassay method for the detection of antibodies comprising:
a) providing a recombinant protein comprising from N-terminus to C-terminus:
a peptidic secretion signal,
a 6-methylguanine-DNA-methyltransferase enzyme (MGMT) or mutant thereof, wherein said MGMT enzyme or MGMT mutant is the protein of SEQ ID NO:2, or a homologous sequence thereof that is at least 80% identical to SEQ ID NO:2 or at least 75% identical to SEQ ID NO:4, and
a protein of a microorganism;

b) contacting the recombinant protein with a biological sample containing antibodies; and
c) detecting whether antibodies in the biological sample bind to the recombinant protein.

2. The immunoassay method of claim 1 , wherein said MGMT enzyme or MGMT mutant is a homologous sequence thereof that is at least 90% identical to SEQ ID NO:2.

3. The immunoassay method of claim 1 , wherein said MGMT enzyme or MGMT enzyme mutant is encoded by the DNA sequence of SEQ ID NO:1, SEQ ID NO:47, or SEQ ID NO:67.

4. The immunoassay method of claim 1 , wherein the protein of a microorganism is a bacterial or viral protein.

5. The immunoassay method of claim 1 , wherein the microorganism is Dengue virus.

6. The immunoassay method of claim 1 , wherein the microorganism is Japanese encephalitis (JE) virus.

7. The immunoassay method of claim 1 , wherein the microorganism is Tick-borne encephalitis (TBE) virus.

8. The immunoassay method of claim 1 , wherein the microorganism is Yellow fever (YF) virus.

9. The immunoassay method of claim 1 , wherein the microorganism is Usutu (USU) virus.

10. The immunoassay method of claim 1 , wherein the microorganism is Rocio virus.

11. The immunoassay method of claim 1 , wherein the microorganism is Murray Encephalitis (MVE) virus.

12. The immunoassay method of claim 1 , wherein the microorganism is Wesselbron (WSL) virus.

13. The immunoassay method of claim 1 , wherein the microorganism is Zika virus.

14. The immunoassay method of claim 1 , wherein the microorganism is West Nile (WN) virus.

15. The immunoassay method of claim 1 , wherein the microorganism is Rift Valley Fever (RVF) virus.

16. The immunoassay method of claim 1 , wherein the microorganism is Toscana (TOS) virus.

17. The immunoassay method of claim 1 , wherein the microorganism is Chikungunya virus.

18. The immunoassay method of claim 1 , wherein the protein of a microorganism is selected from EDIII protein from Dengue, Japanese encephalitis (JE), Tick-borne encephalitis (TBE), Yellow fever (YF), Usutu (USU), Rocio, Murray Encephalitis (MVE), Saint-Louis encephalitis virus, Wesselbron (WSL), Zika and West Nile (WN) viruses.

19. The immunoassay method of claim 1 , wherein the protein of a microorganism is selected from the nucleoprotein N from Rift Valley Fever (RVF) and Toscana (TOS) viruses.

20. The immunoassay method of claim 1 , wherein the protein of a microorganism is selected from 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.

21. The immunoassay method of claim 1 , wherein the biological sample is blood or serum.

Assignees

• Pasteur Institut

Inventors

• Despres Philippe
• Paulous Sylvie
• Crublet Elodie

Classifications

• C12Y201/01063

  Methylated-DNA-[protein]-cysteine S-methyltransferase (2.1.1.63), i.e. O6-methylguanine-DNA methyltransferase · CPC title

• C12N2800/107

  for mammalian · CPC title

• C12N2800/105

  for insects · CPC title

• C12N2015/8518

  expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles · CPC title

• C12N15/85

  for animal cells · CPC title