Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1.

Autor: Pakdaman SF; Department of Biology, Hakim Sabzevari University, Sabzevar, Iran., Vatandoost J; Department of Biology, Hakim Sabzevari University, Sabzevar, Iran., Bos MHA; Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
Jazyk: angličtina
Zdroj: Biotechnology progress [Biotechnol Prog] 2020 Mar; Vol. 36 (2), pp. e2938. Date of Electronic Publication: 2019 Nov 15.
DOI: 10.1002/btpr.2938
Abstrakt: 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 expression and activity by enhancement of γ-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 γ-carboxylation that is mediated by the enzymes γ-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 γ-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 γ-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 γ-carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.
(© 2019 American Institute of Chemical Engineers.)
Databáze: MEDLINE
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