Expression, cDNA cloning, and characterization of the antibacterial peptide cecropin D fromAgrius convolvuli

Cecropins are basic antibacterial peptides that have potent activities against microorganisms. We have cloned and characterized a cecropin-like peptide of the lepidopteran insect Agrius convolvuli and analyzed its expression in Escherichia coli. The full-length cDNA of A. convolvuli cecropin D3 (AcCec) was 318 bp, containing a 5′ untranslated region (UTR) of 47 bp, a 3′ UTR of 82 bp with a poly (A) tail, and an open reading frame (ORF) of 189 bp encoding a polypeptide of 63 amino acids, including a 24 amino acid signal sequence and a 38 amino acid mature peptide (GenBank accession no. GQ888768). The mature peptide is highly similar to D-type cecropin. To understand the effect of C-terminal amidation, while overcoming the disadvantage of its lack in the prokaryote system, we added a lysine residue to AcCec (AcCec-K) and compared its antibacterial activity to the purified AcCec. The recombinant AcCec (rAcCec) and AcCec-K were expressed, respectively, in E. coli Rosetta cells using a pGEX-4T-1 expression vector, which contained the glutathione S-transferase (GST) gene for fusion partner, and the fusion proteins were induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The recombinant proteins were purified by fast protein liquid chromatography (FPLC) using GSTrap FF and Resource RPC column. The result of the inhibition zone suggests that C-terminal lysine residue could increase the activity due to activated phosphorylation.