The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC50 = 13-15 μg/ml).

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.