Expression of recombinant snake venom metalloproteinase disintegrin domain from green pit viper, trimeresurus albolabris, and characterization of its effects on human platelets

Abstract

Disintegrins are involved in inhibition of platelet aggregation. They contain RGD or KGD sequences that can inhibit integrin-mediated cell-cell interactions and platelet aggregation. Therefore, snake venom disintegrins are helpful in therapy of arterial thrombosis and tumor metastasis, and producing an antibody for development of a venom diagnostic test kit. The aim of this thesis is to analyze the full-length sequence of 035 SVM from the cDNA library of Trimeresurus albolabris venom gland and characterize functions of the disintegrin domain of 035 SVM on human platelets. From the primary library, partial cDNA of 035 SVM was obtained. Using 5'-RACE method, the full-length sequence of 035 SVM was derived. Analysis of the nucleotide sequence found that the full-length sequence of 035 SVM mRNA was 2,040 bp in length. It was a type N-II snake venom metalloproteinase with 484 amino acid residues comprising signal region, pro-peptide, metalloproteinase domain, spacer region and disintegrin domain. The nucleotide sequence of 035 SVM showed 84% amino acid sequence identical to trimeresurus jerdonii jerdonitin. The disintegrin domain of 035 SVM, composed of 76 amino acids with a KGD sequence, was expressed in Pichia pastoris and purified using affinity chromatography. The yield of recombinant 035 disintegrin was 3.3 mg/liter of culture medium with molecular weight of approximately 11 kDa and 22 kDa on SDS-PAGE in reduced and non-reduced state, respectively. Recombinant 035 disintegrin was a homodimer that inhibited collagen-induced platelet aggregation with IC[subscript 50] of 976 nM