Expression And Characterization Of Phosphohpases A₂from Venom Of Snake Trimeresurus Stejnegeri

Phospholipase A2(PLA2. EC3.1.1.4) specifically hydrolysis of fatty acid ester bonds at position2of to produce free fatty acids and lysophospholipids. PLA2display several physiology activities, so widely involved in the metabolism and andregulation of human cells. Because of different49th, amino acids, venom PLA2include of D49PLA2and K49PLA2. D49PLA2contains a calcium binding loop, mostly have a strong enzyme activity. K49PLA2is a basic protein without a calcium-binding loop, mostly shows weak enzyme activity. Calcium is a necessary cofactor of PLA2playing enzyme activity, and it is important to pharmacologically actives, but the mechanism is not yet clear. Therefore, the study of calcium ions on the structure and function of PLA2is defiant and attractive. Trimeresurus Stejnegeri phospholipase A2TS-G6D49was selected as modle in this study. The calcium binding affinity and calcium binding structure were studied to show the role of calcium in structure and function of PLA2.TS-G6D49was expressed as a fusion protein EDDIE-TS-G6D49. First, recombinant cloning plasmid pMD18-T-EDDIE-TS-G6D49and the recombinant expression plasmid pET-30a-EDDIE-TS-G6D49were constructed. The EDDIE-TS-G6D49was expressed in inclusion bodies. But the protein did not fold well in the refolding process. Then the recombinant cloning vector pMD18-T-TS-G6D49and recombinant expression plasmid pET-30a-TS-G6D49were constructed. The pET-30a-TS-G6D4was expressed in E. coli BL21(DE3) by induction of1mM IPTG. The protein with14kDa was expressed by SDS-PAGE analysis. The inclusion bodies were prepared by sonication and Triton X-100washsion. The protein was denatured by8M urea and refoldied by the urea concentration gradient dialysis. The protein was purified by HPLC and identified by SDS-PAGE. The enzyme activity was detected by the method of determination of the fatty acids of the hydrolyzate, and the results showed that the specific activity of TS-G6D49was0.3814μmol×min-1×mg-1.TS-G6D49belongs to the secretory PLA2. The enzyme activity site consists of residues of H48, Y52, Y73and D99, the calcium binding ring consists of residues of Y28, G30, G32, and the D49. In order to study the influence of calcium ions on the structure and function of PLA2, five mutants, M-1(Tyr28Asn), M-2(Asp49Lys), M-3(Tyr28Asn, Asp49Lys), M-4(Asp49Asn) and M-5(Tyr28Asn, Gly30Ala, Asp49Asn) were designed. The mutated recombinant cloning vectors were constructed by site-directed mutagenesis based on the recombinant expression plasmid pET-30a-TS-G6D49. The mutans were expressed in E. coli BL21(DE3) by induction of1mM IPTG and detected by SDS-PAGE. The calcium binding affinity and calcium binding structure will be studied to show the role of calcium in structure and function of PLA2.