title: Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli.
abstract: Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H2 O2 . Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Fan
authors.forename: Holger
authors.initials: H
authors.lastname: Schäfer
authors.forename: Jürgen
authors.initials: J
authors.lastname: Reichling
authors.forename: Michael
authors.initials: M
authors.lastname: Wink
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006461:Hemolysis; D006801:Humans; D029069:Impatiens; D008826:Microbial Sensitivity Tests; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D012756:Sheep / Q000097:blood
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; C504508:Ib-AMP4 peptide, Impatiens balsamina; D011993:Recombinant Fusion Proteins
keywords: Bactericidal properties; Drug resistance; Ib-AMP4; Plant antimicrobial peptide; Recombinant production