Molecular cloning and characterization of cecropin from the housefly ( Musca domestica), and its expression in Escherichia coli

Abstract

A cDNA encoding housefly ( Musca domestica) cecropin transcript was isolated from total RNA using RT-PCR, 3′RACE, and λgt11 cDNA library screening, and was expressed in Escherichia coli. This is the first report of a cecropin nucleotide and amino acid sequence in the housefly. The open reading frame of Md-Cec (189 bp) encodes a precursor of 63 aa, which is comprised of a 23 aa signal peptide and a 40 aa mature peptide. In terms of amino acid sequence, Md-Cec shares a high degree of identity (74–82%) with those of some Diptera insects. Northern blot, RT-PCR and in situ hybridization analyses revealed that the prececropin was temporally expressed 5 h after bacteria-challenge in larvae, and was induced in the fat body, epithelia of the body wall, and the epidermis of the midgut. The DNA fragment encoding mature Md-Cec was then subcloned into the pGEX-4T-1 expression vector and was highly expressed in E. coli BL21 with IPTG induction. The expressed proteins, fused to glutathion S-transferase, were purified by glutathion-Sepharose 4B affinity chromatography and cleaved with thrombin, followed by gel filtration chromatography. Recombinant Md-Cec exhibited antimicrobial activity against E. coli.