Expression and purification of high purity soluble chimeric protein VEGI~+ / 第二军医大学学报

ABSTRACT

Objective:To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI~+,so as to lay a basis for studying its biological activity.Methods:Chimeric molecule VEGI~+ was constructed by grafting oligopeptide CTTH- WGFTLC to extraeellular region of VEGI(VEGI_(23-174)).Before ligation into pET30a(+)expression vector,PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing,then pET30a-VEGI was used to transfect BL21(modified E.coli strain).The chimeric protein was purified by metal affinity chro- matography.Western blotting and coomassie blue staining were used for protein identification.Results:The chimeric molecule VEGI~+ was confirmed by restriction enzyme digestion and DNA sequencing.The constructed pET30a-VEGI was confirmed by enzymatic digestion.The expression was mainly in the form of inclusion body.SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23000,with a purity of about 90%.Conclusion:We have successfully constructed the recom- binant plasmid pET30a-VEGI~+ and expressed it in E.coll.And we have obtained high purity of soluble chimeric protein VEGI~+ through affinity chromatography.