Methods and compositions for producing vitamin K dependent proteins

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 NaHPO4, 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).

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 NaHPO4, 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).

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 NaHPO4, 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).

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 NaHPO4, 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.

A host cell comprising a recombinant nucleic acid encoding Factor IX and a recombinant nucleic acid encoding vitamin K epoxide reductase (VKOR), 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 NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C. and wash conditions of 0.1×S SC/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.