Large scale purification of factor X by hydrophobic chromatography.
Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.
Friedberg
R C
RC
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Pizzo
S V
SV
D002845:Chromatography / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005170:Factor X / Q000302:isolation & purification*; D006801:Humans; D007457:Iodine Radioisotopes; D010949:Plasma / Q000032:analysis
D007457:Iodine Radioisotopes; D005170:Factor X