Abstract

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.

Abbreviation

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.