Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their... Show moreReplacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of gamma-carboxylation might be of interest. The expression and functional activity of vitamin K-dependent (VKD) coagulation proteins rely, in part, on the VKD process of gamma-carboxylation that is mediated by the enzymes gamma-carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX-producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT-PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assay and coagulation test. In addition, to quantify gamma-carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX-producing HEK cells, resulting in a 3.2-fold higher expression of functional FIX, which displayed a 1.4-fold enhanced specific activity. Moreover, a 3.9-fold enhanced recovery of fully gamma-carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of gamma-carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs. Show less
Schie, R.M.F. van; Khedr, N. el; Verhoef, T.I.; Teichert, M.; Stricker, B.H.; Hofman, A.; ... ; Visser, L.E. 2012
