Innovating recombinant production of short peptide targeted antimicrobial peptides utilizing clean purification in plants and E. coli bioreactors.

Abstract

Antibiotic-resistant pathogens continue to become a pressing issue globally and current broad spectrum therapies fail to meet this challenge. Therapeutics equipped with targeting moieties would alleviate this issue without facilitating the rise of opportunistic pathogens, and employing small antimicrobial peptides (AMPs) would avoid resistant strains. Fusion of the two could be produced recombinantly at commercial levels in bioreactors such as plants with a purification tag, but issues with phytotoxicity and unexplained low recombinant AMP yield from plant tissue are major limiting factors.

This dissertation addresses the issues of the plant expression platform for AMPs as well as properly assessing the selectivity of a previously discovered 12mer targeting domain (A12C) specific to Staphylococci. To understand why AMPs are poorly expressed in plant expressions systems, meta-analysis of peptide databases was performed and revealed plant-derived AMPs are less cationic in net charge compared to AMPs from organisms like animals and fungi. Using the elastin-like polypeptide as a tag for increased recombinant production and clean temperature shift purification, a survey of AMPs falling under the cysteine-stabilized motif ranging in net charge were produced in Nicotiana benthamiana. It was observed that only the anionic fusions were expressed, and at record levels as high as 563 µg/gram fresh leaf weight with retained antimicrobial activity only pre-protease cleavage. This phenomenon of activity maintained while still fused was studied in E. coli, and it was found the size of the ELP fusion partner dictates antimicrobial potential.

For quicker turnover to facilitate targeting studies, cationic AMPs plectasin and eurocin were expressed in E. coli with the SUMO solubility tag. The targeting domain was assessed for antimicrobial selectivity against a panel of gram positive bacterium. Unexpectedly, there was no enhancement of activity against target bacterium, but a significant decrease in antimicrobial activity against non-target genus.

These studies elucidate that the peptide net charge dictates recombinant AMP expression in plants, and a genus-specific targeting domain can be derived from as small peptide sparing commensal non-target bacteria. Together, these discoveries provide the foundation for the inexpensive production of targeted AMPs in plant bioreactors or in transgenic seed for poultry or livestock.