Calixarene-assisted protein refolding via liquid-liquid extraction.
Calixarene-assisted protein refolding via liquid-liquid extraction.
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DOI：
10.1021/bm070418q
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发表时间：
2007-08
影响因子：
6.2
通讯作者：
K. Shimojo;T. Oshima;H. Naganawa;M. Goto
中科院分区：
文献类型：
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作者：
K. Shimojo;T. Oshima;H. Naganawa;M. Goto
在本文中，我们报告了使用杯芳烃通过液-液转移技术进行蛋白质重折叠。我们发现杯[6]芳乙酸衍生物与尿素变性的细胞色素c在油水界面形成超分子复合物，从而能够通过质子交换机制将蛋白质从8 M尿素水溶液定量转移到有机相中。变性细胞色素c与变性剂完全分离，并与其他变性细胞色素c分子分离，以抑制由于蛋白质-蛋白质相互作用而产生聚集体。在酸性条件下使用适量的 1-丁醇成功地从有机相中回收细胞色素 c。 UV-vis、CD 和荧光光谱表征表明，转移到不含变性剂的水溶液中的细胞色素 c 恢复了其天然结构。使用抗坏血酸重折叠细胞色素 c 的还原动力学表明，该蛋白作为电子转移蛋白提供约 72% 的天然活性。
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.