Molecular cloning and expression of factor v activator (RVV-V) from russell's viper venom

Abstract

Snake venoms contain various types of enzyme such as phospholipase A2, metalloproteinases, serine proteases and L-amino acid oxidases, while non enzymatic proteins including C-types lectins and disintegrins are also found. Daboia russelli siamensis or Russell's viper (RV) is a medically important snake in the central rice-growing area of Thailand. Bitten by such snake causes variety of signs and symptoms associated with severe reduction of blood coagulation factor. The aim of this study is (1) to clone and characterize the factor V activator (RVV-V) cDNA from Russell’s viper gland cDNA library, and (2) to produce the recombinant RVV-V in microorganisms through genetic engineering approach. RVV-V is a member of serine protienase family, consisting of the catalytic triad containing active site, 12 cysteins performing 6 disulfide bonds and one N-glycosylation site. The 5’ segment of the RVV-V cDNA was obtained from the cDNA library by 5’-RACE. The nucleotide amino acid sequences of RVV-V were analyzed by molecular biology softwares. In addition, two novel serine proteinases cDNA, designed RVAF and RVBF, were also cloned. Since only amino acid sequence of RVV-Vγ was reported, cDNAs of the RVV-V, RVAF anf RVBF from this study have been submitted in GenBank. In addition, to improve our understanding of the phylogenetic relationship, the amino acid sequences of mature proteins of the RVV-V, RVAF, RVBF and other snake venom serine proteinases were used to construct of phylogenetic tree, a useful tool for classification of structure and function of proteins and DNAs. The mature protein-encoding sequence of RVV-V was cloned to produce the recombinant protein (rRVV-V) in bacteria and yeast. To express rRVV-V in E. coli, the DNA fragment was subcloned in to the pET32a vector, the expression vector that enhances solubility of the recombinant protein, by including the thioredoxin tag with the expressed recombinant protein. In addition, the rRVV-V was also co-expressed with molecular chaperone. The solubilized rRVV-V was observed in the E. coli cell lysate. However, the rRVV-V had no activity on the chromogenic substrate. The effort to improve the expression system had moved to the yeast expression system. Picia pastoris, a methylotrophic yeast that allows production of post-translational modified recombinant. Two expression vectors for P. pastoris were used, pPIC-Z and pPink. However, expression of rRVV-V was unsuccessful in both vectors. The rRVV-V could not be observed as shown by SDS-PAGE analysis, and protease activity on chromogenic substrate was not found. Therefore, improvement of recombinant protein expression system is required in further study to produce the functional rRVV-V for further applications.