Prokaryotic Expression Vector Construction And Expression, Purification And Identification Of Apoptin

Objective:To construct prokaryotic expression vector for improved TAT-VP3 fusion gene, express the target proteins and purify it, removal of GST-tag, and finally get the apoptin protein.Methods:Design and amplify improved TAT-VP3 fusion gene by PCR and clone into expression vector, construct prokaryotic expression vector for improved TAT-VP3 fusion gene, and transform the constructed recombinant plasmid to E.coli BL21(DE3)plyS for expression under induction of IPTG. Identify the expressed product by 15% SDS-PAGE. Express the improved TAT-VP3 fusion protein under induction of temperature by IPTG.The expressed product was purified by GST affinity chromatography and identified by SDS-PAGE and Western bolt. Removal the GST-tag of purified fusion protein by Prescission protease.Results:The prokaryotic expression vector for improved TAT-VP3 fusion gene was constructed successfully, Which was corroborated by restriction analysis and sequencing. Improved TAT-VP3 fusion protein was stably expressed under induction of temperature. With relative molecular masses of 42000, was expressed. The expressed product was purified successfully by GST affinity chromatography and showed good specipicity as proved by Western bolt. Removal the GST-tag of purified fusion protein by Prescission protease, finally get the apoptin protein.Conclusion:The prokaryotic expression vector for improved TAT-VP3 fusion gene was constructed successfully, the target protein was expressed and purified, which laid a foundation of further study on its immunogenicity and antitumor activity, Removal of GST-tag, and finally has been get the apoptin protein.