Human Hexastatin genetic optimization, protein expression, purification and preliminary application
10.3760/cma.j.issn.1673-4181.2012.02.010
- VernacularTitle:人Hexastatin基因的优化、蛋白的表达纯化及初步应用
- Author:
Xiao TANG
;
Naling SONG
;
Xin HE
;
Yueying WANG
;
Qian LIU
;
Lei WEN
;
Dezhi WANG
;
Ying HAN
;
Heng ZHANG
- Publication Type:Journal Article
- Keywords:
Hexastatin protein;
Expression and purification;
MTT activity examination
- From:
International Journal of Biomedical Engineering
2012;35(2):103-107,后插6
- CountryChina
- Language:Chinese
-
Abstract:
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.
