Modification, expression, and protein purification of the recombinant cecropin D-like antibacterial peptide derived from haemolymph of Agrius convolvuli (sweet potato hornworm) larvae

Cecropin D is one of the three major cecropins that displays a high degree of homology with cecropins A and B characterized by a blocked C terminus, but lacks the N-terminal lysine and contains two glycine residues. In this study, we investigated the cecropin D-like antibacterial peptide from the immunized Agrius convolvuli larvae. The 3' and the 5' regions of Agrius convolvuli cecropin (AC cec) gene were amplified using PCR protocol for the rapid amplification of cDNA ends (RACE). The recombinant AC cec was expressed in E. coli Rosetta cells for pGEX 4T-1 expression vector. AC cec gene was amplified with forward primer AC cec BamHI, reverse primer 1 AC cec XhoI (ori-cec), and reverse primer 2 AC cec + Lys XhoI (cec-K). The expressed proteins, fused to GST, were purified by glutathione-Sepharose 4B affinity chromatography and cleaved by thrombin, followed by Resourse reverse phase chromatography. Additionally, inhibition zone assay was performed to test antimicrobial activities of ori-cec and two modified recombinant cecropins, with a lysine extension at the C terminus (cec-K) and with a Gly-Ser-Pro-Glu-Phe pentapeptide extension at the N terminus (GSPEF-cec), against gram-positive [G(+)] and gram-negative [G(-)] bacteria. The study of inhibition mechanism revealed that while cec-K possessed an increased broad spectrum of bactericidal activity, GSPEF-cec exhibited a reduced activity against G(-) but retained activity against G(+) bacteria.