Prokaryotic expression and action mechanism of antimicrobial LsGRP1 recombinant protein containing a fusion partner of small ubiquitin-like modifier.

Antimicrobial peptides (AMPs) are peptides exhibiting broad-spectrum antimicrobial activities and considered as potential therapeutic agents. LsGRP1, a novel AMP derived from defense-related LsGRP1 protein of Lilium, was proven to inhibit kinds of bacteria and fungi via alteration of microbial membrane permeability and induction of fungal programmed cell death-like phenomena by in vitro assays using synthetic LsGRP1. In this study, the prokaryotic production of LsGRP1 recombinant protein containing an N-terminal fusion partner of the yeast small ubiquitin-like modifier (SUMO) was achieved by using optimized Escherichia coli host and purification buffer system, which lead to a high yield of soluble SUMO-LsGRP1 fusion protein. In vitro assay revealed that E. coli-expressed SUMO-LsGRP1 exhibited even better antifungal activity as compared to synthetic LsGRP1. Meanwhile, the ability of SUMO-LsGRP1 in conducting fungal membrane permeabilization and programmed cell death was verified by SYTOX Green staining and 4',6-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick-end labeling assays, respectively, indicating that E. coli-expressed SUMO-LsGRP1 shares identical modes of action with synthetic LsGRP1. Herein, this E. coli expression system enables the effective and convenient production of antimicrobial LsGRP1 in a form of SUMO-fused recombinant protein.