Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli

Abstract

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the beta (1-4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc-hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc-hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc-hly may have important potential application as a future safely administered human drug and food additive.

Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli

“…Microbicidal assay was performed as previously described (Ueno et al 2010;Lu et al 2010). Each bacterial strain was cultured to exponential phase and diluted 1,000 times with LB culture medium.…”

“…To address this issue, numerous biological expression systems have been established to produce AMPs in hosts such as prokaryotic cells and yeast cells (Jin et al 2006;Xu et al 2007;Chen et al 2009;Lu et al 2010). However, each of these systems presents their own set of problems.…”

Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

“…Evidences of synergism between lysozyme and cecropin have generated interest to design novel antimicrobial protein with a broader spectrum of activity and increased potency [23]. Therefore, it is necessary to efficiently express recombinant antimicrobial peptide parasin I fused with human lysozyme in yeast expression system.…”

Effects of <i>Musca domestica</i> Cecropin on the Adhesion and Migration of Human Hepatocellular Carcinoma BEL-7402 Cells

“…1,2) They are able to form specific amphipathic alpha-helices, which allow them to target nonpolar lipid cell membranes, and have strong antibiotic activity against both Gram-positive and Gram-negative bacteria at low micromolar concentrations. 3,4) More recent studies have also indicated the antitumor activity of cecropins against various cancer cell lines of leukemia, lymphoma, colon carcinoma, small cell lung cancer and gastric cancer, but without damage to human normal cells which would make it a good candidate for the development of anti-tumor agents. 5,6) The Musca domestica gene cecropin (GenBank accession number: EF175878) was cloned in our laboratory from the larva.…”