Expression, purification, and characterization of recombinant human factor X.
A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the -2 residue (Thr-->Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure-function relationships by mutational analysis.
Rudolph
A E
AE
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110-8118, USA.
Mullane
M P
MP
Porche-Sorbet
R
R
Miletich
J P
JP
D000990:Antithrombin III / Q000494:pharmacology; D001777:Blood Coagulation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005170:Factor X / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014162:Transfection
D065427:Factor Xa Inhibitors; D010743:Phospholipids; D011498:Protein Precursors; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000990:Antithrombin III; D005170:Factor X; D015951:Factor Xa