Making plants into cost-effective bioreactors for highly active antimicrobial peptides.

• Antimicrobial peptides (AMPs) native to plants tend to be less cationic. • Expression in Nicotiana benthamiana only successful with anionic AMPs. • AMPs fused to elastin-like polypeptide were active even without protease cleavage. • Antimicrobial activity was 10x greater than previously reported for AMPs. As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants than in E. coli expression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. This suggests that there may be a physical characteristic of the previously described heterologous AMPs which impedes efficient expression in plants. Using a meta-analysis of protein databases, this study has determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. To apply this finding to plant expression, the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to -5, was tested in the tobacco, Nicotiana benthamiana. Elastin-like polypeptide (ELP) was used as the carrier protein for AMP expression. ELP fusion allowed for a simple, cost-effective temperature shift purification. Using this system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 μg/gfw). Furthermore, antimicrobial activity against Staphylococcus epidermidis was an order of magnitude greater (average minimum inhibitory concentration MIC of 0.26μM) than that typically seen for AMPs expressed in E. coli systems and was associated with the uncleaved fusion peptide. In summary, this study describes a means of expressing AMP fusions in plants in high yield, purified by a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity and without the need for a peptide cleavage step. [ABSTRACT FROM AUTHOR]