Characterization of recombinant plectasin: Solubility, antimicrobial activity and factors that affect its activity

Plectasin is the first known fungal defensin with potent activity against Gram-positive bacteria. To evaluate the potential therapeutic application of plectasin, we produced plectasin and investigated its solubility, activity and factors that affect its antimicrobial activity. Recombinant plectasin was produced from Escherichia coli in high yield by integration of fusion expression and on-column cleavage. Including 0.5 M arginine significantly increased the solubility of plectasin in acetic acid buffer with 10% glycerol from 89 μg/ml to 408 μg/ml. Plectasin was soluble at 846 μg/ml in Tris-glycerol-EDTA buffer. Plectasin was active against Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus with minimum inhibitory concentrations of 2 and 0.5 μg/ml. Much lower or no activity was observed toward Gram-negative bacteria and fungi. Plectasin (128 μg/ml) did not exhibit hemolytic activity toward rabbit erythrocytes. The activity of plectasin toward S. aureus was decreased by reduction with dithiothreitol, indicating that the disulfide-bond is essential for maximal activity. Plectasin was bactericidal under physiological concentrations of mono- and divalent cations. This activity was markedly attenuated by divalent cations in a concentration-dependent manner, however, with complete inhibition occurring at Ca2+ concentrations greater than 25 mM. These results suggested that the presence of the disulfide-bond and the absence of divalent cations play key roles in the antimicrobial activity of plectasin.