Calixarene-assisted protein refolding via liquid-liquid extraction

Kojiro Shimojo, Tatsuya Oshima, Hirochika Naganawa, Masahiro Goto

    Research output: Contribution to journalArticlepeer-review

    23 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)3061-3066
    Number of pages6
    Issue number10
    Publication statusPublished - Oct 2007

    All Science Journal Classification (ASJC) codes

    • Bioengineering
    • Biomaterials
    • Polymers and Plastics
    • Materials Chemistry


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