Enhanced functional recombinant factor VII production by HEK 293 cells stably transfected with VKORC1 where the gamma-carboxylase inhibitor calumenin is stably suppressed by shRNA transfection.
BACKGROUND
Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo gamma-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational gamma-carboxylation, the recovery of fully gamma-carboxylated and functional proteins is low.


METHODS
In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K gamma-carboxylase cofactor and in addition stably silenced the gamma-carboxylase inhibitory protein calumenin.


CONCLUSIONS
Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C.
Wajih
Nadeem
N
Department of Internal Medicine, Wake Forest University, Winston-Salem, North Carolina, USA.
Owen
John
J
Wallin
Reidar
R
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D005167:Factor VII / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007668:Kidney / Q000166:cytology; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics*; D014162:Transfection / Q000379:methods; D064417:Vitamin K Epoxide Reductases
C510527:CALU protein, human; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D005167:Factor VII; D006899:Mixed Function Oxygenases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases