Biocharacterization of a recombinant thrombin-like enzyme from Gloydius shedaoensis, in Escherichia coli

摘要

Snake venom proteolytic enzymes attract special interest due to their effects on hemostatic system and in turn to substantially help the pharmaceutical industry in the search for new drugs. In this study, a snake venom thrombin-like enzyme termed gloshedobin was produced in an active and soluble form by Escherichia coli and purified to homogeneity as judged by SDS-PAGE analysis. The recombinant gloshedobin, which includes a NusA solubility fusion tag, exhibited amidolytic activity toward N-α -benzoyl-DL-arginine p-nitroanilide (BApNA) and fibrinogenolytic activity toward bovine fibrinogen. Like most natural thrombin-like enzymes, the recombinant gloshedobin preferentially cleaved bovine fibrinogen's towards BApNA was inhibited by serine protease inhibitors (PMSF and TPCK), consistent with its assignment to the serine protease family. The thrombin inhibitor heparin, disulfide reductants (dithiothreitol and β -mercaptoethanol), as well as the metal chelator EDTA, did not affect this enzyme's amidolytic activity. If fibrinogen was the substrate, some inhibitors behaved in a different manner: 1) none of the serine protease inhibitors except PMSF had any effect, and 2) EDTA hampered the activity,suggesting that metal ion(s), in particular transition metal ions, play a role in the fibrinogenolytic process. These results showed gloshedobin's unique enzymatic properties compared with other thrombin-like enzymes, and might provide additional insights of use in applying recombinant fibrinogenolytic enzymes in the clinical treatment of thrombosis diseases.