title: Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.
abstract: Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human β-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human β-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.
authors.affiliation: Institute of Feed Science, Zhejiang University, Key Laboratory of Animal Nutrition and Feed Science, Ministry of Agriculture (East China), Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Hangzhou 310058, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Luan
authors.forename: Yong Gang
authors.initials: YG
authors.lastname: Xie
authors.forename: Yu Tian
authors.initials: YT
authors.lastname: Pu
authors.forename: Hai Wen
authors.initials: HW
authors.lastname: Zhang
authors.forename: Fei Fei
authors.initials: FF
authors.lastname: Han
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
authors.forename: Yi Zhen
authors.initials: YZ
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002176:Candida albicans / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D047668:Inteins; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014325:Tromethamine / Q000031:analogs & derivatives; D023083:beta-Defensins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D054804:Cathelicidins
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; C106641:DEFB4A protein, human; D010455:Peptides; D011994:Recombinant Proteins; D023083:beta-Defensins; C080383:peptide 18A; D014325:Tromethamine; C026272:Bistris; D054804:Cathelicidins