Fusion expression and high-level preparation of a glycine-rich antibacterial peptide (SK66) derived from Drosophila in Escherichia coli

Hui-juan Liu, Zhi-guo Feng, Jun Lang, Yan-jiao Li, Guang-yuan He and Zheng-wang Chen.

Abstract：SK66, a derivative of the gene cg13551 of Drosophila containing 66 amino acid peptide with N-terminal serine and C-terminal lysine, shows high antimicrobial activities. To obtain it in large amounts, the mature DNA fragment of SK66 was acquired from the pMD18-T-SK66 simple vector using PCR and then inserted into the Nco I and Xho I enzyme-cutting sites of pET-32a plasmid, the recombinant vector named pET-32a-SK66 was transformed into the competent cell E. coli BL 21. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 44% of the total proteins). Then the expressed product was purified by affinity binding chromatography with Ni-NTA,salt-out, freeze-dried. The SK66 was cleaved from the fusion protein by enterokinase, purified by using RP-HPLC and has strong antibacterial activity.

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