The protease from the latex of Calotropis procera was isolated by an aqueous two-phase system (ATPS). The systems consist of polyethylene glycol (PEG 4000. 6000 and 8000) at concentrations of 9, 12 and 15% (w/w) with salts (Na-citrate, MgSO4, K2HPO4, and (NH4)(2)SO4) at concentrations of 11, 14 and 17% (w/w) were investigated. The highest protease recovery was found in the PEG-rich phase of the system, comprising of 12% PEG 4000-17% MgSO4. For optimization of the system to obtain the higher yield of protease, the system pH (4, 7 and 10) or NaCl addition (2, 4 and 6%, w/w) was studied. At acidic (pH 4.0) and alkaline (9.0) conditions of the systems the reduction of K-E and protease recovery was clearly observed compared to that of the neutral pH (7.0). The addition of NaCl up to a final concentration of 6% (w/w) significantly increased the yield to 107% of the control. Molecular weight distribution and activity staining showed that the isolated protease had the molecular weight of similar to 38 kDa. However, the isolated protease had no activity under reducing condition (beta ME). Under cathodic electrophoresis, protease from C. procera showed the same protein pattern to purified papain. (C) 2010 Elsevier Ltd. All rights reserved.