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.
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.
Wajih
Nadeem
N
Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Hutson
Susan M
SM
Owen
John
J
Wallin
Reidar
R
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014812:Vitamin K / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases
D011994:Recombinant Proteins; D014812:Vitamin K; D005164:Factor IX; D006899:Mixed Function Oxygenases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases