Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag

Antimicrobial peptides (AMPs), such as the linear amphipathic cathelicidins, are produced widely in the natural world and are active against a broad range of pathogenic microorganisms. Their potential as a new range of antibiotics has prompted numerous studies of AMP structure and function. Most such studies are performed with chemically synthesised peptides, but a simple and rapid biosynthetic route would offer a more cost-effective alternative for the production of AMPs and analysis of their structure/function relationships. The cysteine protease domain (CPD) from Vibrio cholerae MARTX toxin possesses an autocleaving ability that is inducible by inositol hexakisphosphate (IP6). When coupled with a hexa-histidine tag and fused to the C-terminus of an AMP, this AMP–CPD fusion may be expressed in Escherichia coli and purified using immobilized metal affinity chromatography. A brief on-column induction of cleavage liberates the AMP, and subsequent polishing using hydrophobic interaction resin allows for purification of the peptide within a day. We used this system to express and purify several 18-residue cathelicidin variants and tested their activity on E. coli, Pseudomonas putida, Bacillus subtilis and Candida albicans. This approach to linear AMP production may aid rapid construction and purification of structural variants for subsequent functional analysis.

The pET28-CPD expression vector and AMP purification protocol. (A) The desired AMP coding sequence may be inserted upstream of the CPD domain between the NcoI and BamHI sites. The start codon is indicated in bold. The codon immediately preceding the BamHI site must code for a leucine to allow CPD cleavage to occur. (B) Schematic of the AMP–CPD purification protocol.Figure optionsDownload as PowerPoint slideHighlights
► The production of antimicrobial peptides (AMPs) by a simple, rapid and cost-effective biosynthetic route is described.
► Linear cathelicidin AMPs are produced as recombinant proteins, fused to an inducible, self-cleaving protease (CPD) tag.
► AMPs are purified by IMAC, on-column cleavage and a final polishing step using hydrophobic interaction resin within a single day.
► The recombinant system allows rapid production of variant AMP sequences for functional studies.