Expression of Pleurocidin from winter flounder in Escherichia coli and optimization of culture conditions.
- Author:
Xuejiao XU
;
Xiangdong ZHA
;
Yuanyuan CHE
;
Lijuan MA
;
Siqun WU
;
Peilong YANG
;
Huoqing HUANG
;
Bin YAO
- Publication Type:Journal Article
- MeSH:
Animals;
Cloning, Molecular;
Escherichia coli;
metabolism;
Fish Proteins;
biosynthesis;
Flounder;
Recombinant Fusion Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2016;32(3):365-374
- CountryChina
- Language:Chinese
-
Abstract:
To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
