Methods and compositions for producing vitamin K dependent proteins

Patent metadata

Field	Value
Publication number	US-9441208-B2
Application number	US-201414490244-A
Country	US
Kind code	B2
Filing date	Sep 18, 2014
Priority date	Sep 23, 2003
Publication date	Sep 13, 2016
Grant date	Sep 13, 2016

Abstract

The present invention relates to methods and compositions for improving the productivity of recombinant vitamin K dependent protein expression in host cells.

First claim

That which is claimed is:

1. A cell that contains and expresses a recombinant nucleic acid comprising a nucleic acid encoding vitamin K epoxide reductase (VKOR) operatively associated with a heterologous promoter, wherein the recombinant nucleic acid encoding 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), 1 mM 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.

2. A method for expressing a vitamin K dependent protein in a host cell, comprising the steps of:
(a) introducing into a host cell a nucleic acid encoding a vitamin K dependent protein;
(b) introducing into the host cell a recombinant nucleic acid encoding a vitamin K epoxide reductase (VKOR) and a recombinant nucleic acid encoding a vitamin K dependent carboxylase, wherein the recombinant nucleic acid encoding 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), 1 mM 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
(c) expressing the nucleic acids of steps (a) and (b).

3. A method for expressing a vitamin K dependent protein in a host cell, comprising the steps of:
(a) providing a host cell that expresses a nucleic acid encoding a vitamin K dependent protein;
(b) introducing a recombinant nucleic acid coding for a vitamin K epoxide reductase (VKOR) into the host cell and a recombinant nucleic acid encoding a vitamin K dependent carboxylase, 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), 1 mM 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
(c) expressing the nucleic acids of steps (a) and (b).

4. A method for expressing a vitamin K dependent protein in a host cell, comprising the steps of:
(a) providing a host cell that expresses a heterologous nucleic acid encoding a vitamin K epoxide reductase (VKOR), wherein the recombinant nucleic acid encoding 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), 1 mM 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;
(b) introducing a nucleic acid coding for a vitamin K dependent protein into the host cell and a nucleic acid encoding a vitamin K dependent carboxylase; and
(c) expressing the nucleic acids of steps (a) and (b).

5. The method of claim 2 , wherein the nucleic acid encoding the vitamin K dependent protein is selected from the group comprising factor VII, factor IX, factor X, prothrombin, Protein C and Protein S.

6. A cell that contains and expresses a recombinant nucleic acid comprising a nucleic acid encoding vitamin K epoxide reductase (VKOR) operatively associated with a heterologous promoter, wherein the recombinant nucleic acid encoding 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), 1 mM 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

wherein said expressed VKOR converts vitamin K epoxide to vitamin K.

7. The cell of claim 6 , wherein said expressed VKOR is warfarin sensitive.

8. The cell of claim 6 , further expressing a nucleic acid encoding a vitamin K dependent protein and expressing a nucleic acid encoding vitamin K dependent carboxylase.

9. The cell of claim 8 , wherein the nucleic acid encoding a vitamin K dependent protein is a heterologous nucleic acid.

10. The cell of claim 8 , wherein the nucleic acid encoding vitamin K dependent carboxylase is a heterologous nucleic acid.

11. The cell of claim 8 , wherein the vitamin K dependent protein is selected from the group c

Assignees

• Univ North Carolina Chapel Hill

Inventors

• Stafford Darrel W
• Li Tao

Classifications

• C12N9/64

  derived from animal tissue · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Y304/21006

  Coagulation factor Xa (3.4.21.6) · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title