RECONSTITUTION OF CATALYTICALLY COMPETENT HUMAN ZETA-THROMBIN BY COMBINATION OF ZETA-THROMBIN RESIDUES A1-36 AND B1-148 AND AN ESCHERICHIA-COLI EXPRESSED POLYPEPTIDE CORRESPONDING TO ZETA-THROMBIN RESIDUES B149-259

Human zeta-thrombin, a catalytically competent serine proteinase, arises from a single chymotryptic cleavage at Trp-148 in alpha-thrombin to generate two noncovalently associated polypeptide segments designated zeta-1-thrombin (the 36-residue A-chain disulfide linked to B-chain residues B1-148) and zeta-2-thrombin (B149-259). We report here the expression of recombinant zeta-2-thrombin in Escherichia coli and the reconstitution of catalytically competent zeta-thrombin by combination of zeta-1-thrombin with recombinant zeta-2-thrombin. A DNA fragment encoding zeta-2-thrombin was cloned into a pATH2 expression vector as a trpE-zeta-2 fusion gene, in which a factor Xa cleavage site was inserted between the trpE and the zeta-2-thrombin gene. High-level expression of this fusion protein was achieved under the control of the E. coli trp promoter. The expressed zeta-2-thrombin was liberated from the fusion protein by factor Xa cleavage, reduced with DTT, and purified to homogeneity by reverse-phase HPLC. Oxidation of the reduced zeta-2-thrombin in the presence of 80-mu-M CuSO4 and 6 M urea at pH 8.15 yielded material that was indistinguishable on HPLC from zeta-2-thrombin isolated by resolution of human zeta-thrombin. Catalytically active zeta-thrombin was generated by combination of recombinant zeta-2-thrombin with zeta-1-thrombin that was isolated by resolution of human zeta-thrombin. Recombinant zeta-thrombin displayed catalytic activities, toward a small chromogenic substrate and fibrinogen, that were similar to those of alpha-thrombin prepared from human blood plasma and zeta-thrombin obtained by treatment of alpha-thrombin with chymotrypsin. This result indicates that the information for formation of a catalytically competent conformation resides in the primary structure of zeta-thrombin and suggests that studies of variants of zeta-thrombin produced by site-directed mutagenesis of zeta-2-thrombin could facilitate identification of the structural and functional determinants of the interactions of thrombin that are important in blood coagulation.