Abstract
Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.
Highlights
Recombinant factors VII, VIIa, VIII, IX, and protein C have become important pharmaceuticals in treatment of traumatic bleeding complications (1), hemophilia (2), and sepsis (3)
We have shown that overexpression of VKORC1 in baby hamster kidney (BHK) cells increases the capacity of the cell’s ␥-carboxylation system (14)
Together these experiments strongly suggested that the tandem chromatography procedure outlined in Fig. 1 provided 1) the “tool” we needed to purify functional, fully ␥-carboxylated recombinant human factor IX (r-human factor IX (hFIX)) and 2) an in situ assay to measure the capacity of our cell lines to produce functional, fully ␥-carboxylated variants of r-hFIX among total r-hFIX produced by the cells
Summary
Gla, ␥-carboxyglutamic acid; r-hFIX, recombinant human factor IX; VKOR, vitamin K 2,3-epoxide reductase; VKORC1, a putative subunit of VKOR (12); ER, endoplasmic reticulum; BHK, baby hamster kidney; TBS, Tris-buffered saline; HPLC, high performance liquid chromatography. VKORC1 Increased Production of Functional Recombinant FIX fected with a VKORC1 cDNA construct, produce 2.9-fold more fully ␥-carboxylated functional r-hFIX than r-hFIX-producing BHK cells using the endogenous vitamin K-dependent system of the cell to ␥-carboxylate the protein. This finding provides new opportunities for increased production of fully ␥-carboxylated and functional recombinant vitamin K-dependent proteins
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.