Secretory ranalexin produced in recombinant Pichia pastoris exhibits additive or synergistic bactericidal activity when used in combination with polymyxin B or linezolid against multi-drug resistant bacteria.

Ranalexin, a cationic peptide from frogs, is a potent therapeutic antimicrobial peptide (AMP). Its limited availability is an obstacle for a wider application. A high-level production of AMPs via bioengineering is possible but remains a challenging task. In the current study, we investigated the potential antibacterial properties of recombinant ranalexin, expressed in the yeast Pichia pastoris. A 78-bp DNA fragment encoding the mature ranalexin peptide with a 6-His tag on its C-terminus was designed using the preferred codon usage of P. pastoris. The gene was inserted into pPICZaA and transformed into competent cells of P. pastoris strain KM71. The yield of secretory ranalexin reached up to ~6 mg/L culture. Time-kill curve analysis of ranalexin against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) demonstrated a concentration- dependent rapid bactericidal activity. In checkerboard assays, the combinations of ranalexin with the established antibiotics polymyxin B or linezolid reduced the MIC additively in most tested bacteria. Time-kill assays indicated a significant synergism in E. coli and MRSA when ranalexin was used in combination with antibiotics, even at concentrations of 1/4 MIC or 1/2 MIC of ranalexin, respectively. Thus we propose that secretory ranalexin produced in P. pastoris could be a useful tool to unravel ranalexin’s biological function and for use in future in vivo studies against multi- resistant bacterial infections.