Expression, purification and preliminary identification of Melittin in E. Coli
- VernacularTitle:蜂毒肽基因在大肠杆菌中的表达、纯化及初步鉴定
- Author:
Wenhe ZHU
;
Liling TIAN
;
Miaonan SUN
;
Dejun SUN
- Publication Type:Journal Article
- Keywords:
Melittin;
e. coli;
expression;
purification
- From:
Chinese Journal of Biochemical Pharmaceutics
2010;31(1):1-5
- CountryChina
- Language:Chinese
-
Abstract:
Purpose Melittin was expressed in prokaryotic vector pGEX-2T for production. Methods Melittin gene synthesized with enterokinase digested sequence,the gene was cloned into vector pGEX-2T,and constructed a recombinant plasmid of pGEX-MEL. Then the recombinant vector was introduced into E. coli BL21 (DE3)for expression. Fusion protein was purified by affinity chromatography. Hemolytic activity of Melittin was detected. Results Analysis result showed that the expression products accumulate in the cells to about 29.5 % of total cell protein. Detection of western blot using ant-GST as the first antibody showed that a special blot was revealed among the expression products. It certified that we have succeeded in expressing the fusion protein. SDS-PAGE showed that most part of the products is resoluble. The purity of obtained protein is 95% , by through GST affinity chromatography system. Melittin is harvested with a recovery of 80% by EK digestion. Test results showed melittin has good hemolytic activity. Conclusion We have expressed Melittin successfully by prokaryotic expression system.
