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 mu g/ml to 408 mu g/ml. Plectasin was soluble at 846 mu g/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 mu g/ml. Much lower or no activity was observed toward Gram-negative bacteria and fungi. Plectasin (128 mu g/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 Ca(2+) 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. (C) 2011 Elsevier Ltd. All rights reserved.