Biocharacterization of a recombinant thrombin-like enzyme from Gloydius shedaoensis, in Escherichia coli
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 fibrinogens 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 enzymes 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 gloshedobins 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.
anticoagulant defibrinogenation serine protease thrombin-like enzyme inhibitor
Jianqiang Xu Qing Yang Wimal Ubhayasekera Sherry L. Mowbray Jan-Christer Janson
Department of Bioscience and Biotechnology, Dalian University of Technology,No.2 Linggong Road, Dali Department of Molecular Biology, Swedish University of Agricultural Sciences,Biomedical Center, Upps Department of Surface Biotechnology, Uppsala University, Biomedical Center, P.O.Box 577, Uppsala 751
国际会议
The 12th Asian Pacific Confederation of Chemical Engineering Congress(第十二届亚太化工联盟大会暨化工展览会)
大连
英文
2008-08-04（万方平台首次上网日期，不代表论文的发表时间）