Background: To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a\r\ncost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of\r\nthe N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The\r\nexpression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombininlike\r\npeptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs\r\nwere purified and evaluated for antimicrobial activity.\r\nResults: Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene\r\nwas then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then\r\nexpressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in\r\nthe cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in\r\nvitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving\r\nAMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All\r\nrecombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.\r\nConclusions: The method described in this report allows the fast synthesis of genes that are optimized for overexpression\r\nin E. coli and for the production of sufficiently large amounts of peptides for functional and structural\r\ncharacterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is\r\na low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic\r\nanalyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/\r\nHis-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.
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