Expression vector and methods of producing high levels of proteins

Patent metadata

Field	Value
Publication number	US-9260721-B2
Application number	US-201213471501-A
Country	US
Kind code	B2
Filing date	May 15, 2012
Priority date	Jun 20, 2005
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

A process for high expression of protein of interest using an expression vector. The process comprises at least the following regulatory elements:
a) a CMV promoter, or a functional variant thereof, b) an intron, c) TPL or a functional variant thereof, d) VA gene or a functional variant thereof, and e) a bovine growth hormone polyadenylation sequence or a functional variant thereof.

First claim

We claim:

1. A mammalian expression vector which consists of:
a) a CMV promoter, or a functional variant thereof,
b) a chimeric intron having the nucleic acid sequence of SEQ ID NO: 1,
c) tripartite leader element (TPL) or a functional variant thereof,
d) a viral associated (VA) RNA gene or a functional variant thereof, and
e) a bovine growth hormone polyadenylation sequence or a functional variant thereof; and
f) a nucleic acid sequence encoding a protein of interest.

2. The vector as claimed in claim 1 , wherein the TPL is a TPL having the nucleic acid sequence of SEQ ID NO: 4.

3. The vector as claimed in claim 1 , wherein the VA gene or functional variant thereof has the nucleic acid sequence genes are having a sequence of SEQ ID NO: 5.

4. The vector as claimed in claim 1 , further comprising a selection and amplification marker selected from the group consisting of dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase, asparagine synthetase, and glutamine synthetase, or a functional variant thereof.

5. The vector as claimed in claim 1 , encoding the gene for erythropoietin.

6. The vector as claimed in claim 1 , encoding the gene for fusion protein, TNFR-IgGFc.

7. The vector as claimed in claim 1 , encoding a gene for rituximab, trastuzumab, bevacuzumab or other monoclonal antibodies.

8. The vector as claimed in claim 1 , encoding one of more genes capable of expression in a mammalian cell.

9. A mammalian cell transformed with an expression vector as claimed in claim 1 .

10. The mammalian cell as claimed in claim 9 wherein the mammalian cell is selected from the group consisting of Cos, CHO, CHO DHFR−, BHK1, and NS0.

11. A mammalian expression vector consisting of:
a) a CMV promoter, or a functional variant thereof,
b) a chimeric intron having the nucleic acid sequence of SEQ ID NO: 1,
c) a tripartite leader element (TPL) having the nucleic acid sequence of SEQ ID NO: 4 or a functional variant thereof,
d) a viral associated (VA) VA genes an RNA gene having the nucleic acid sequence of SEQ ID NO: 5 or a functional variant thereof,
e) a bovine growth hormone polyadenylation sequence or a functional variant thereof; and
f) a nucleic acid sequence encoding a protein of interest.

12. The vector as claimed in claim 11 , encoding the gene for erythropoietin.

13. The vector as claimed in claim 11 , encoding the gene for fusion protein, TNFR-IgGFc.

14. The vector as claimed in claim 11 , further comprising a selection and amplification marker selected from the group consisting of dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase, asparagine synthetase, and glutamine synthetase, or a functional variant thereof.

Assignees

• Singh Arun K
• Goel Ashish
• Mendiratta Sanjeev K
• Cadila Healthcare Ltd

Inventors

• Singh Arun K
• Goel Ashish
• Mendiratta Sanjeev K

Classifications

• C12N15/85 Primary

  for animal cells · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/7151

  for tumor necrosis factor [TNF], for lymphotoxin [LT] · CPC title

• C12N2840/60

  from viruses · CPC title

• C12N2710/16122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title