Antitumor19Peptide Induced Expression In E.Coli And Activity Of Verification

Antitumor19peptide Tumstatin near the C-terminal amino acids185-203.The function ofit is direct inhibition of tumor cell proliferation and promote tumor cell apoptosis.Antitumor19peptide was extracted from animal costs higher and synthetic less.With the development ofmolecular biology techniques.The use of engineering bacteria to produce recombinant antitumor19peptide will become a development trend in the future.The study transformed recombinant vector pet32a-19peptides into four kinds of E. coliBL21(DE3)plysS、BL21(DE3)、BL21and Rosetta(DE3).The recombinant protein was inducedexpression with IPTG and identified by SDS-PAGE.The results of Gel electrophoresis wasanalyzed by1D Gel Image Processing Software.A new band of about22kD was appeared,thesize of band was consisten with the expectation. The results showed that the target gene wasexpressed in recombinant bacteria，and the E. coli BL21(DE3) strain as highest expressionwhich concentration was18mg/L,named pET32a-19peptide-BL21(DE3),which as a follow-upexperiment engineered bacteria.The pET32a-19peptide-BL21(DE3) was carried out15generations subculture.Thechange in expression of recombinant protein was not significant. The results showed that theheredity of plasmid is steady. Respectively, to determine optimal fermentation conditions ofrecombinant bacteria by single factor and orthogonal experiment. The optimum fermentationconditions was determined by orthogonal experiment is:the pH value of medium is7.0,inoculumis3%,concentration of IPTG is0.1mmol/L, OD600=0.7,temperature is41℃and the inductiontime is5h. The expression value of the recombinant protein is54.57mg/L.The bacteria was broken by supersonic lysis, the supernate and deposition aftercentrifugation was detected by SDS-PAGE. The results showed that the recombinant proteinwas expressed through inclusion. The recombinant protein was purified through the method ofaffinity chromatography by Ni Sepharose6Fast Flow.The purity was analysed bySDS-PAGE,which reached more than95%.The recombinant protein was refold by gradient dialysis, lyophilized the purifiedprotein.The activity experiment was conducted by MTT. The inhibition rate of tumor cellsreached70%when the concentration of recombinant protein was200μg/mL.