Human Hexastatin genetic optimization, protein expression, purification and preliminary application / 国际生物医学工程杂志

ObjectiveTo optimize human Hexastatin gene,to express,purify protein and conduct activity experimental research,and to provide a theoretical basis for further study of Hexastatin.MethodsHuman Hexastatin gene was optimized and synthesized.It was connected to the pET28a expression vector,induced to express by isopropyl β-D-1-thiogalactopyranoside(IPTG),and optimized induction conditions.After the ultrasonication of bacterial cells and inclusion bodies,the recombinant fusion protein was purified with Ni-NTA chromatographic column,analyzed and identified by SDS-PAGE and Western Blot,and conduct activity experimental research in vitro by MTT.ResultsConstructed production was pET28a-Hexastatin expression plasmid.The human Hexastatin protein was expressed in E.coli BL21 the high level and accounted for 45.1％ of the total bacterial protein.The purification of recombinant protein purified with Ni-NTA chromatographic column reached 90％,and the concentration was 80 μg/ml.Human Hexastatin protein can restrain the growth of C6,MCF-7 and human vascular endothelial cell (HMEC) cells,and inhibition ratio reach to 72.9％±3.6％,48.8％±2.9％,52.7％±2.5％,respectively through MTT test.ConclusionThe optimized human Hexastatin protein was expressed successfully,which confirmed the inhibition to tumour cells.It is a new way for anti-angiogenesis therapy of tumour.

Expression and purification of soluble recombinant Hexastatin in E.coli / 中国生化药物杂志

purpose To construct the expression vector of Hexastatin gene,to express and to purify the recombinant protein for further activity research.Methods The human Hexastatin gene was isolated by RT-PCR from EC9706 cells total RNA and cloned into pMDl8-T for sequencing.Then the Hexastatin gene was subcloned into pMAL-c4x expression vector and induced to express by IPTG.The recombinant fusion protein was purified with Amylose Resin Heads.Results RT-PCR product was about 687 bp and its sequence was the same as that of Hexastatin reported.The recombinant protein was expressed in E.coli BL21 with high level and the soluble protein accounted for 24.8% of the total bacterial protein,The purification of recombinant protein purified with Amylose Resin Heads reached more than 90%.Conclusion The cloning,expression and purification of human Hexastatin have laid a foundation for its anti-angiogenesis therapy for tumor.