Sequence Analysis And Expression Of Chitribrisin, A Thrombin-like Enzyme From The Venom Glands Of Chinese Green Pit Viper (trimeresurus Albolabris), In Pichia Pastoris

Snake venoms contain various kinds of enzymes which affected hemostasisand thrombosis. It was demonstrated that most of snake venom thrombin-like enzymes (SVTLEs) did not active factor XIII in vivo, and could cleave fibrinogen to non-cross-linked fibrin releasing fibrinopeptide A (FPA) or fibrinopeptide B (FPB).Compared with thrombin , SVTLEs can cleavae fibrinogen in vitro,making it into fibrin, resulting in blood clotting; in vivo, SVTLEs can consume thrombin-fibrinogen, reduce blood viscosity, from the aspects of blood rheology microcirculation, which plays the role of antithrombotic has therefore applied in the clinical treatment of thrombosis and other diseases. At the moment, the SVTLEs preparations which being medically used are extracts from snake venoms and encounter problems in purity and cost-effectiveness, but those problems would be solved by gene engineering.In this study, two primers were designed with reference to the known cDNA sequences of SVTLEs, especially those from green pit viper venom. Total RNA was obtained from venom glands of T. albolabris which captured in Chongqing, as were amplified by one step RT-PCR, product of about 805 bp by was obtained and cloned into pMD18-T. Sequencing results proved to be a novel TLE cDNA,apart from the primers, the length of encoding cDNA was 777 bp. Assaying the nucleotide sequence of the cDNA allowed the complete amino acid sequence forchitribrisin to be determined. Chitribrisin was previously expressed in Escherichia coli in our laboratory, but the formation of inclusion bodies resulted in loss of biological activity of the recombinant chitribrisin.Then the mature gene of chitribrisin was subcloned into the vector pPICZαA, an P. pastoris expression vector based on AOX 1 promoter. Having been induced by methanol for 96 hours, the recombinant enzyme was examined in the cytoplasm by SDS—PAGE analysis,After purification by HiTrap Heparin HP column, the purified recombinant gave a 52 KD band on SDS-PAGE gel. Then produced 46 mg target protein in 960 ml supernatant. The results showed that the biological activity of the expression is much more high that the product of E. coli. And it was also determined by fibrinogen clotting time assay and fibrinogenolytic activity assay chitribrisin showed fibrinogen clotting activity and a fibrinogenolytic activity against theαandβchains, but did not cleave itsγchain, the results were consistent with the expression of E. coli.