Recovery of refolded aggregated proteins from ionic liquids via biphasic systems

We demonstrate that the refolding and activity recovery of aggregated proteins can be achieved simply and efficiently using two-phase systems of hydrophobic ionic liquids (ILs)/buffer. The high solubility of thermally aggregated concanavalin A (Con A) was observed in the hydrated tetra-n-octylammonium dihydrogen phosphate ([N8888][dhp]). Upon adding a buffer solution, the two-phase separation of the IL and buffer was confirmed, with the upper layer consisting of the IL phase and the lower layer consisting of the aqueous phase. Dissolved Con A was transferred to the buffer phase. Transfer efficiency of Con A into the buffer phase increased as the water content of the IL phase decreased, which was influenced by the buffer concentration and temperature. Con A transferred into the buffer phase exhibited a recovery of sugar chain-binding activity to levels comparable to that of native Con A.