Full Length Research Paper

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

Hui-juan Liu^1#, Zhi-guo Feng^1,2#, Jun Lang¹, Yan-jiao Li¹, Guang-yuan He¹* and Zheng-wang Chen¹*

¹Key Laboratory of Molecular Biophysics Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.

²College of Life Sciences, Xinyang Normal University, Xinyang Henan 464000, China.

*Corresponding author. E-mail: zwchen@mail.hust.edu.cn. Fax: +86 27 87792024.

#These authors contributed equally to this paper

Abbreviations: SK₆₆, 66-residue mature peptide with N-terminal serine and C-terminal lysine; IPTG, isopropyl β-D-thiogalactoside; LB, luria-bertini; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; EK, enterokinase; RP-HPLC, reverse-phase high performance liquid chromatography.

Accepted 24 July, 2009

SK_66, 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.

Key words: Antibacterial peptides, Drosophila melanogaster, fusion expression, preparation, SK_66.