Analgesic Activity Of The Scorpion Peptide (as) Expression Product In Vitro Refolding

The protein drugs prepared by expression of heterogenous gene in E.coli have been one of important methods to produce genetic engineering pharmaceuticals, but the recombinant proteins are accumulated as inclusion bodies in E.coli. How to refold proteins at high concentration with high yield is one of critical techniques of recombinant proteins production. Renauration of recombinant BmK AS inclusion bodies was studied for promoting to solve the problem.First, the gene of protein AS was cloned into pET28a, constructing the expression plasmid pET28a-AS,under induction of IPTG ,using SDS-PAGE analysis, the most of protein AS existed in the supernatant, the level of expression was 31%; the gene of protein AS was cloned into pET28a, constructing the expression plasmid pET28a-His-AS,under induction of IPTG ,using SDS-PAGE analysis, the most of protein AS existed in the supernatant, the level of expression was 35%.Then, the bacterial pellets were harvested and subjected to ultrasonication. After centrifugation ,the pellets containing the inclusion bodies of AS were washed with Triton X-100 buffer and 1M Gu-HCl. After denaturing the inclusion bodies in 8M Gu-HCl, the concentration of denaturing proteins was 0.1mg/ml using step-by-step dilution, and the yield were 22.6% and 23.2%.At last, after protein renauration, the protein was purified by chromatography. The protein with expression plasmid pET28a-AS was purified by SP Sepharose Fast Flow chromatography. The protein content was tested using the Bradford method。Finally , per liter ferment liquid could gain protein 1.6 mg; The protein with expression plasmid pET28a-His-AS was purified by Chealting Sepharose 4B Fast Flow chromatography . The protein content was tested using the Bradford method。Finally,per liter ferment liquid could gain protein 1.8 mg.