Construction of fusion expression vector pET22b-SUMO-FGFR4 and optimization of expression conditions in E.coli
10.13481/j.1671-587x.20160402
- VernacularTitle:融合表达载体 pET22b-SUMO-FGFR4的构建及其在大肠杆菌中表达条件的优化
- Author:
Wei LIU
;
Yang YAO
;
Xiaoxiao MA
;
Yuxuan DENG
;
Di MEI
;
Lei LIU
;
Huiyan WANG
- Publication Type:Journal Article
- Keywords:
small ubiquitin-like modifier;
fibroblast growth factor receptor 4;
recombinant fusion proteins
- From:
Journal of Jilin University(Medicine Edition)
2016;42(4):642-647
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To design the small ubiquitin modification-fibroblast growth factor receptor 4 (SUMO-FGFR4) fusion gene and construct the expression vector pET22b-SUMO-FGFR4, to optimize the expression conditions. Methods:The SUMO-FGFR4 fusion gene was obtained by Overlap PCR and was connected to pET22b;the recombinant expression vector pET22b-SUMO-FGFR4 was obtained. The influence of lactose concentration, induction time,induction temperature,induction point and adding mode of lactose in the expression levels was observed,and the best induction condition was determined; then the solubility of recombinant protein was analyzed.Results:The SUMO-FGFR4 fusion protein was highly expressed,the molecular weight of the fusion protein was about 40 000 and it could bind with FGFR4 specific antibody.When the lactose concentration was 1.0 g·L-1 ,the induction time was 3 h,the induction temperature was 37℃,the value of A (600)was 0.8,the expression level was highest;but adding mode of lactose had no remarkable effect on the protein expression.The expression level of recombinant protein induced by lactose was higher than IPTG.SUMO-FGFR4 protein existed in a form of inclusion body.Conclusion:The SUMO-FGFR4 fusion protein is expressed successfully in this study while lactose is used as inducer and the best expression conditions are confirmed.
