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

doi:10.13481/j.1671-587x.20160402

Abstract

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.

Key words: small ubiquitin-like modifier, fibroblast growth factor receptor 4, recombinant fusion proteins

CLC Number:

LIU Wei, YAO Yang, MA Xiaoxiao, DENG Yuxuan, MEI Di, LIU Lei, WANG Huiyan. Construction of fusion expression vector pET22b-SUMO-FGFR4 and optimization of expression conditions in E.coli[J].Journal of Jilin University Medicine Edition, 2016, 42(04): 642-647.

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Construction of fusion expression vector pET22b-SUMO-FGFR4 and optimization of expression conditions in E.coli

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