Optimization of expression and purification of SUMO‐tagged LL‐37 peptides

Antimicrobial peptides (AMPs) are a class of naturally occurring biomolecules found in all multicellular organisms, and are used as a first line of defense for plants and animals. AMPs have been found to cause bacterial membrane rupture and in some cases they also inhibit intracellular function, in both cases resulting in bacterial lysis and death. A broad collection of AMPs have been discovered that can selectively target bacteria, yeasts, fungi, viruses, and even cancer cells. For example, AMPs that target bacteria are typically positively charged, and therefore interact via electrostatic interactions with negatively charged bacterial membranes. Cathelicidins, are small, cationic, amphiphilic AMPs found in many mammalian species. Cathelicidins exibit a broad range of antimicrobial activity against fungi, bacteria, and enveloped viruses, allowing for innate immunity in the skin. LL-37 is the sole member of the human cathelicidins. Numerous studies have investigated the structure and function of LL-37, in an effort to develop antimicrobial therapeutics based on its core structure. Many of these potential therapeutics rely on automated peptide synthesis. We seek to contribute to the understanding of the LL-37 peptide structure and function by further investigating key residues which are required for antimicrobial activity, and optimize the expression of recombinant LL-37-based peptides in E.coli. A structure-based design strategy informed the construction of a series of mutant LL-37 peptides, that were generated using site-directed mutagenesis. The following mutants were investigated: a series of single amino acid substitutions, G14P, K12R, K18R, K25R, addition of a hydrophobic tail (LLAA) and hydrophilic tail (LRQA). Cloning was achieved using the ThermoFisher Champion pET SUMO Protein Expression System, and sequences were confirmed using Sanger sequencing. The 11-kD SUMO (small ubiquitin-related modifier) on the C-terminus of the recombinant protein enhances the expression and solubility of the LL-37 peptide in E. coli. The His-tagged, LL-37-SUMO peptides are expressed in BL21(DE3) E. coli, and following removal of the SUMO and His tags, ongoing studies examining the activity of the recombinant peptides against gram-positive and gram-negative pathogens continues.