Eepression and characterization of a recombinant novel snake venom metalloproteinase from green pit viper (Cryptelytrop albolabris)

Abstract

Snake venom metaloproteases (SVMPs) may degrade extracellular matrix and/or clotting factors, as well as inhibit integrin and platelet functions, studying them not only gives US deeper insights in pathogenesis of snakebites, but also potentially yields novel antiplatelet agents. A clone of novel RGD-containing, P-ll type, snake venom metalloproteinase was isolated from the green pit viper, C. albolabris, venom gland cDNA library. Sequence analysis revealed that it belonged to P-llb subclass of SVMP and 81% identical to Jerdonitin from Trimeresurus jerdonii. They contained 2 conserved cysteines forming a disulfide bond, which held metalloproteinase and disintegrin together in mature proteins. The N-terminally histidine-tagged construct of metalloproteinase and disintegrin domains was inserted into the pPICZ α A vector and expressed in Pichia pastoris. The recombinant protein was approximately 32kD on Western blot probed with anti-polyhistidine antibody. The recombinant SVMP dose dependently inhibited platelet aggregation induced by collagen with the IC₅₀ of 5.6 µM. . However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism should be through blocking of collagen receptor(s). This recombinant protein has a potential to be a novel anti-platelet agent.