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 nonconvalently 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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gan
Z R
ZR
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Lewis
S D
SD
Stone
J R
JR
Shafer
J A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009876:Operon; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D013917:Thrombin