EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN FACTOR-X

A system is described for producing recombinant factor X with properti es very similar to human plasma factor X, Optimization of the expressi on system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney c ells (BHK cells) for the expression of functional factor X, It was als o determined that production of factor X by 293 cells requires the sub stitution of the -2 residue (Thr --> Arg) which affords the removal of the factor X propeptide, Purification of recombinant and plasma facto r X is accomplished using a calcium-dependent monoclonal antibody dire cted against the gla domain, The proteins are comparable by sodium dod ecyl sulfate polyacrylamide gel electrophoresis, The rate and extent o f activation by the factor X coagulant protein from Russell's viper ve nom and by factors Ma and VIIIa are similar; activation of the recombi nant 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 leave the same apparent affinity for the activated platelet surface as judged by their abilit y to activate prothrombin, Finally, inhibition by antithrombin, with o r without heparin, and inhibition by the tissue factor pathway inhibit or are equivalent, Recombinant factor X produced by this method is the refore well suited for probing structure-function relationships by mut ational analysis, (C) 1997 Academic Press.