An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin

Human recombinant prethrombin-2 was produced in Escherichia coli. The expre ssed prethrombin-2 formed intracellular inclusion bodies from which the pro tein was refolded by a simple one-step dilution process in buffer consistin g of 50 mM Tris-HCl, containing 20 mM CaCl2 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin- based COOH-terminal peptide affinity chromatography, and then activated wit h Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha -thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fi brinogen, activation of protein C, and thrombin-activatable fibrinolysis in hibitor, reactivity with antithrombin, clotting activity, and platelet aggr egation. The kinetic data showed no differences in activity between our rec ombinant alpha -thrombin and plasma-derived alpha -thrombin. The yield of r efolded recombinant human prethrombin-2 was about 4-7% of the starting amou nt of solubilized protein. In addition, the final yield of purified refolde d protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.