Abstract
BackgroundTo facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.ResultsTwo or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.ConclusionsThe method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.
Highlights
To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs
Recombinant AMPs expressed in E. coli often include 1-2 non-native amino acid residuals at the N terminus of the target protein due to a specific linker sequence recognized by endoproteases or chemical agents, typically located between the native protein sequence and the tag [18]
Construction of his-EDDIE-GFP fusion expression plasmid The construction of the pET30a/His-EDDIE-GFP vector was based on pET30a/His- EDDIE-Cecropin AD (CAD)
Summary
To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. Recombinant AMPs expressed in E. coli often include 1-2 non-native amino acid residuals at the N terminus of the target protein due to a specific linker sequence recognized by endoproteases or chemical agents, typically located between the native protein sequence and the tag [18] These non-authentic N termini of AMPs often alter their characteristics. The N-terminal autoprotease, Npro, of classical swine fever virus (CSFV) cleaves itself between the C-terminal Cys168 and position 169 [19], which represent the authentic N-terminal amino acid of the target protein It is a relatively hydrophobic protein that tends to form insoluble aggregates on refolding, preventing autoproteolysis [20]. The fusion protein EDDIE-CAD was successfully expressed in E. coli after codon optimization and the purified recombinant mature Cecropin AD (CAD) was fully bioactive [22]
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