Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP intein tag.
An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa MAP 2, a polyalanine AMP. Pa MAP 2 was fused to an ELP intein tag where the ELP (Elastin like polypeptide) was used to promote aggregation and fast and cost effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa MAP 2 fused to the N termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa MAP 2 showed a MIC of 25mM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa MAP 2 can be produced in both rich and defined media at yields 50 fold higher than reported for other AMPs produced by the ELP intein system, and in comparable yields to expression systems with protease or chemical cleavage.
Journal of biotechnology