Calixarene-assisted protein refolding via liquid-liquid extraction

In this paper we report on protein refolding by means of a liquid-liquid transfer technique using a calixarene. We have found that a calix[6]areneacetic acid derivative forms a supramolecular complex with urea-denatured cytochrome c at the oil-water interface, which enables quantitative transfer of the protein from an 8 M urea aqueous solution into an organic phase through a proton-exchange mechanism. Denatured cytochrome c is completely separated from the denaturant and is isolated from other denatured cytochrome c molecules to suppress the generation of aggregates due to protein-protein interactions. The recovery of cytochrome c from the organic phase is successfully achieved under acidic conditions using an appropriate amount of 1-butanol. UV-vis, CD, and fluorescence spectroscopic characterizations demonstrate that cytochrome c transferred into a denaturant-free aqueous solution regains its native structure. The reduction kinetics of refolded cytochrome c using ascorbic acid indicates that the protein provides approximately 72% of native activity as an electron-transfer protein.