Production of fully processed and functional factor X in a furin-secreting mammalian expression system
View Patent ↗Disclosed herein are methods for production of fully-processed mature Factor X in an expression system producing a controlled amount of furin between 50 U/mL and 300 U/mL of culture supernatant. Also disclosed are transformed cells, expression systems, and expression vectors for the expression of furin and Factor X.
1. An isolated eukaryotic transformed cell comprising:
a first exogenous nucleic acid encoding a human furin and a second exogenous nucleic acid encoding a human Factor X, such that the transformed cell expresses and secretes the human furin and the human Factor X into a culture supernatant, wherein the human furin is secreted at a concentration of about 60 U/mL to about 300 U/mL in the culture supernatant after culture for about 36 to about 78 hours and at least 85% of the human Factor X is fully processed, and wherein the first exogenous nucleic acid and the second exogenous nucleic acid are present on one or two plasmids.
2. The isolated eukaryotic transformed cell of claim 1 , wherein the plasmid(s) is a non-viral expression vector.
3. The isolated eukaryotic transformed cell of claim 1 , wherein the plasmid(s) is a viral expression vector.
4. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin is secreted at a concentration of about 100 to about 300 U/mL in the culture supernatant after culture for about 36 to about 78 hours.
5. The isolated eukaryotic transformed cell of claim 4 , wherein at least 90% of the human Factor X produced by the transformed cell is fully processed.
6. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin is secreted at a concentration of at least about 90 to about 100 U/mL in the culture supernatant after culture for about 36 to about 78 hours.
7. The isolated eukaryotic transformed cell of claim 6 , wherein at least 95% of the human Factor X produced by the transformed cell is fully processed.
8. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin concentration in the culture supernatant reaches about 60 to about 300 U/mL after culture for about 42 to about 72 hours.
9. The isolated eukaryotic transformed cell of claim 1 , wherein the cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell, a primate kidney cell, a fibroblast, and a mouse myeloma cells.
10. The isolated eukaryotic transformed cell of claim 1 , further comprising an exogenous nucleic acid encoding a human vitamin K epoxide reductase (VKOR).
11. An isolated eukaryotic transformed cell comprising:
a first expression vector comprising an exogenous nucleic acid encoding a human furin; wherein the transformed cell secretes the human furin into the culture supernatant at a concentration of about 60 U/mL to about 300 U/mL after culture for about 36 to about 78 hours, and wherein the first expression vector is a plasmid.
12. The isolated eukaryotic transformed cell of claim 11 , wherein the cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell, a primate kidney cell, a fibroblast, and a mouse myeloma cells.
13. The isolated eukaryotic transformed cell of claim 11 , further comprising an exogenous nucleic acid encoding a human vitamin K epoxide reductase (VKOR).
14. The isolated eukaryotic transformed cell of claim 11 , wherein the transformed cell further comprises an exogenous nucleic acid encoding a protein, and wherein the protein comprises an Arg-(Lys/Arg)-Arg motif and is cleavable by the human furin.
15. The isolated eukaryotic transformed cell of claim 14 , wherein the protein is von Willebrand Factor, Factor II, Factor IX, Factor X, Protein C, Protein S, or Protein Z.
16. The isolated eukaryotic transformed cell of claim 14 , wherein the exogenous nucleic acid encoding the protein is on a second expression vector.