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.
Shimojo
Kojiro
K
Division of Environment and Radiation Sciences, Nuclear Science and Engineering Directorate, Japan Atomic Energy Agency, Tokai-mura, Ibaraki, Japan.
Oshima
Tatsuya
T
Naganawa
Hirochika
H
Goto
Masahiro
M
D001205:Ascorbic Acid / Q000737:chemistry; D047250:Calixarenes / Q000737:chemistry; D002625:Chemistry, Organic / Q000379:methods; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014508:Urea / Q000737:chemistry; D014867:Water / Q000737:chemistry
D014867:Water; D047250:Calixarenes; D014508:Urea; D045304:Cytochromes c; D001205:Ascorbic Acid