BACKGROUNDProtein refolding represents the limiting step in the recovery of these molecules from inclusion bodies and from denaturing operations. This raises the need for establishing different methodologies to efficiently recover and refold proteins. Aqueous two-phase systems (ATPS) besides being a primary recovery operation provide good and affordable physicochemical conditions to refold these molecules. RESULTS48 different PEG-potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase. Invertase in both forms has a partition preference for the bottom salt-rich phase where the polymer molecular weight and the tie-line length (TLL) selected for the system play a preponderant role. Refolding percentages of up to 100% can be obtained in the polymer-rich phase and between 50% and 75% in the bottom phase. CONCLUSIONSThis study reports the characterization of how the different ATPS design parameters such as polymer molecular weight, tie-line length (TLL) and volume ratio (V-R), influence protein partition and refolding using invertase as the protein model. A PEG 3350 g mol(-1), TLL 25.0% w/w and V-R = 1.00 system was selected as the best option for denatured invertase recovery and refolding with an enzyme recovery of 60.8% and a refolding percentage of 59.7% in the bottom phase of the system. These findings represent advancements en route to establishing an ATPS operation for the potential in situ recovery and refolding of denatured protein based-products. (c) 2015 Society of Chemical Industry