Increased Production of Functional Recombinant Human Clotting Factor IX by Baby Hamster Kidney Cells Engineered to Overexpress VKORC1, the Vitamin K 2,3-Epoxide-reducing Enzyme of the Vitamin K Cycle

• Published in: The Journal of biological chemistry 2005-09, Vol.280 (36), p.31603-31607
• Main Authors: Wajih, Nadeem, Hutson, Susan M., Owen, John, Wallin, Reidar
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Chromatography, Affinity; Cricetinae; Electrophoresis, Polyacrylamide Gel; Factor IX - biosynthesis; Factor IX - genetics; Humans; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Transduction, Genetic; Vitamin K - metabolism; Vitamin K Epoxide Reductases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M505373200

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 γ-carboxylation system that converts the proteins to γ-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 γ-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 γ-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 γ-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 γ-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.