title: Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli.
abstract: Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD-intein-pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.
authors.affiliation: State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Xiao-lin
authors.initials: XL
authors.lastname: Meng
authors.forename: Jin-ping
authors.initials: JP
authors.lastname: Xu
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.forename: Chun-wei
authors.initials: CW
authors.lastname: Ma
mesh_terms: D000818:Animals; D054807:Cecropins / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D007915:Lepidoptera / Q000737:chemistry* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D054807:Cecropins; D011993:Recombinant Fusion Proteins