Methods and compositions for producing vitamin K dependent proteins

1. An isolated host cell comprising a recombinant nucleic acid coding for a vitamin K epoxide reductase (VKOR), wherein the recombinant nucleic acid coding for the VKOR comprises a nucleotide sequence selected from the group consisting of:

1) the nucleotide sequence of SEQ ID NO:9;

2) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 10;

3) a nucleotide sequence having at least 95% identity with the nucleotide sequence of SEQ ID NO:9 and that encodes a VKOR;

4) a nucleotide sequence that hybridizes with the complement of the nucleotide sequence of (1) or (2) above under stringent conditions as represented by hybridization conditions of 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65° C. and wash conditions of 0.1×SSC/0.1% SDS at 68° C. and that encodes a VKOR;

5) a nucleotide sequence that encodes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 10 and that encodes a VKOR; and

6) a nucleotide sequence that differs from the nucleotide sequence of(1), (2), (3), (4) or (5) above due to the degeneracy of the genetic code and that encodes a VKOR,

and a recombinant nucleic acid coding for a vitamin K dependent protein, wherein both the recombinant VKOR and the recombinant Vitamin K dependent protein are expressed in said host cell.

2. The host cell of claim 1 , wherein either the nucleic acid coding for recombinant VKOR or the nucleic acid coding for the recombinant Vitamin K dependent protein or both are expressed via an expression mode selected from the group consisting of one or more of the following: induced, transient, and permanent expression.

3. The host cell of claim 1 , wherein the host cell is a mammalian cell.

4. The host cell of claim 3 , wherein the mammalian cell is a cell derived from a mammalian cell line selected from the group consisting of one or more of the following: CHO cells and HEK293 cells.

5. The host cell of claim 1 , wherein the recombinant Vitamin K dependent protein comprises a coagulation factor.

6. The host cell of claim 1 , wherein the coagulation factor is selected from the group consisting of one or more of the following: factor II, factor VII, factor VIIA, factor IX, factor X, prothrombin, Protein C, activated Protein C and Protein S.

7. The host cell of claim 6 , wherein the coagulation factor comprises human factor IX.

8. The host cell of claim 6 , wherein the coagulation factor comprises human factor VII.

9. A cell culture system comprising isolated cells that contain a recombinant nucleic acid coding for a vitamin K epoxide reductase (VKOR), wherein the recombinant nucleic acid coding for the VKOR comprises a nucleotide sequence selected from the group consisting of:

1) the nucleotide sequence of SEQ ID NO:9;

2) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:10;

3) a nucleotide sequence having at least 95% identity with the nucleotide sequence of SEQ ID NO:9 and that encodes a VKOR;

4) a nucleotide sequence that hybridizes with the complement of the nucleotide sequence of (1) or (2) above under stringent conditions as represented by hybridization conditions of 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65° C. and wash conditions of 0.1×SSC/0.1% SDS at 68° C. and that encodes a VKOR;

5) a nucleotide sequence that encodes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 10 and that encodes a VKOR; and

6) a nucleotide sequence that differs from the nucleotide sequence of(1), (2), (3), (4) or (5) above due to the degeneracy of the genetic code and that encodes a VKOR,

and a recombinant nucleic acid coding for a vitamin K dependent protein, wherein both the recombinant VKOR and the recombinant Vitamin K dependent protein are expressed in said cells.

10. The cell culture system of claim 9 , wherein the cultured cells are mammalian cells.

11. The cell culture system of claim 10 , wherein the mammalian cells are selected from the group consisting of one or more of the following: CHO cells and HEK293 cells.

12. The cell culture system of claim 9 , wherein the recombinant Vitamin K dependent protein is a coagulation factor.

13. The cell culture system of claim 12 , wherein the coagulation factor is selected from the group consisting of one or more of the following: factor II, factor VII, factor VIIa, factor IX, factor X, prothrombin, Protein C, activated Protein C and Protein S.

14. The cell culture system of claim 13 , wherein the coagulation factor comprises human factor IX.

15. A method for improving the productivity of recombinant vitamin K dependent protein expression in a host cell, comprising the steps of:

(a) providing the isolated host cell of claim 1 ; and

(b) expressing the recombinant nucleic acids of the host cell of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the host cell.

16. A method for improving the productivity of recombinant vitamin K dependent protein expression in a host cell, comprising the steps of:

(a) providing the isolated host cell of claim 1 , having a recombinant nucleic acid coding for a vitamin K dependent protein integrated into its genome; and

(b) expressing the nucleic acids of the host cell of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the host cell.

17. The method of claim 16 , wherein the recombinant nucleic acid coding for a vitamin K dependent protein is stably expressed.

18. A method for improving the productivity of recombinant vitamin K dependent protein expression in a host cell, comprising the steps of:

(a) providing the isolated host cell of claim 1 , having a recombinant nucleic acid coding for a vitamin K epoxide reductase (VKOR) integrated into its genome; and

(b) expressing the nucleic acids of the host cell of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the host cell.

19. The method of claim 18 , wherein the recombinant nucleic acid coding for VKOR is stably expressed.

20. A method for improving the productivity of recombinant vitamin K dependent protein expression in a cell culture system, comprising the steps of:

(a) providing the cell culture system of claim 9 ; and

(b) expressing the recombinant nucleic acids of the cells of the cell culture system of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the cell culture system.

21. A method for improving the productivity of recombinant vitamin K dependent protein expression in a cell culture system, comprising the steps of:

(a) providing the cell culture system of claim 9 , having a recombinant nucleic acid coding for a vitamin K dependent protein integrated into the genome of the cells of the cell culture system; and

(b) expressing the nucleic acids of the cells of the cell culture system of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the cell culture system.

22. The method of claim 21 , wherein the recombinant nucleic acid coding for a vitamin K dependent protein is stably expressed.

23. A method for improving the productivity of recombinant vitamin K dependent protein expression in a cell culture system, comprising the steps of:

(a) providing the cell culture system of claim 9 , having a recombinant nucleic acid coding for a vitamin K epoxide reductase (VKOR) integrated into the genome of the cells of the cell culture system; and

(b) expressing the nucleic acids of the cells of the cell culture system of step (a), thereby improving the productivity of recombinant vitamin K dependent protein expression in the cell culture system.

24. The method of claim 23 , wherein the recombinant nucleic acid coding for VKOR is stably expressed.