1.Expression of Pleurocidin from winter flounder in Escherichia coli and optimization of culture conditions.
Xuejiao XU ; Xiangdong ZHA ; Yuanyuan CHE ; Lijuan MA ; Siqun WU ; Peilong YANG ; Huoqing HUANG ; Bin YAO
Chinese Journal of Biotechnology 2016;32(3):365-374
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.
Animals
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Cloning, Molecular
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Escherichia coli
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metabolism
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Fish Proteins
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biosynthesis
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Flounder
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Recombinant Fusion Proteins
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biosynthesis