Vectors for protein manufacture
The present invention relates to vectors and their use to develop host cell lines for production of a protein of interest, and in particular to vectors which utilize a weak promoter to drive a selectable marker.
1. A vector for expression of a protein of interest comprising a nucleic acid sequence encoding a selectable marker in operable association with a first promoter sequence and a nucleic acid sequence encoding the protein of interest operably linked to a second promoter sequence, wherein the first promoter sequence is a viral Self-Inactivating (SIN) Long Terminal Repeat (LTR) promoter sequence that is at least 95% identical to SEQ ID NO:3.
2. The vector of claim 1 , wherein the SIN LTR promoter sequence is SEQ ID NO:3.
3. The vector of claim 1 , wherein the selectable marker is Glutamine Synthetase (GS).
4. The vector of claim 1 , wherein the selectable marker is Dihydrofolate Reductase (DHFR).
5. The vector of claim 1 , wherein the vector comprises a single poly A signal sequence in operable association with said selectable marker and said nucleic acid encoding the protein of interest.
6. The vector of claim 1 , wherein the vector comprises a first poly A signal sequence in operable association with said selectable marker and a second poly A signal sequence in operable association with said nucleic acid encoding the protein of interest.
7. The vector of claim 1 , wherein the protein of interest is selected from the group consisting of an Fc-fusion protein, an enzyme, an albumin fusion, a growth factor, a protein receptor, a single chain antibody (scFv), a single chain-Fc (scFv-Fc), a diabody, and minibody (scFv-CH3), Fab, single chain Fab (scFab), an immunoglobulin heavy chain, and an immunoglobulin light chain.
8. The vector of claim 1 , wherein the vector is a retroviral vector.
9. The vector of claim 1 , wherein said vector is a plasmid.
10. A host cell comprising the vector of claim 1 .
11. The host cell of claim 10 , wherein the host cell is a GS knockout cell line.
12. The host cell of claim 11 , wherein the host cell is a DHFR knockout cell.
13. The host cell of claim 10 , wherein the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell and a CAP cell.
14. The host cell of claim 10 , wherein the host cell comprises from about 1 to 1000 copies of the vector.
15. The host cell of claim 10 , wherein the host cell further comprises at least a second vector that encodes and allows for expression of a second protein of interest, and wherein said second vector does not include a selectable marker.
16. The host cell of claim 15 , wherein the first protein of interest in the first vector is one of an immunoglobulin heavy or light chain and the second protein in the second vector is the other of an immunoglobulin heavy or light chain.
17. A host cell culture comprising host cells according to claim 10 .
18. A process for producing a protein of interest comprising culturing host cells according to claim 10 and purifying the protein of interest from the host cell culture.