Goto M, Hashimoto Y, Fujita T, Ono T, Furusaki S
Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.
Biotechnol Prog. 2000 Nov-Dec;16(6):1079-85. doi: 10.1021/bp000073m.
Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.
以变性核糖核酸酶A作为包涵体模型，在异辛烷中用二（2-乙基己基）磺基琥珀酸钠（AOT）配制反胶束进行复性。在这个新型复性过程中，采用固液萃取作为基于液液萃取的普通反胶束蛋白质萃取方法的替代方法。首先，考察了诸如AOT浓度、W(o)（=[H₂O]/[AOT]）和pH等操作参数对固体变性蛋白质溶解到反胶束溶液中的影响。高AOT浓度、高W(o)值和水池中的高pH促进了溶解。这些条件有利于固体蛋白质聚集体在有机溶剂中的分散。其次，通过添加谷胱甘肽作为氧化还原试剂，对溶解在反胶束溶液中的变性核糖核酸酶A进行复性。即使在高蛋白浓度下（在水性介质中通常会发生蛋白质聚集），通过调节氧化还原试剂的组成也能实现核糖核酸酶A的完全复性。此外，通过优化反胶束中的含水量（W(o)）和水池的pH提高了复性速率。最后，通过向反胶束溶液中加入极性有机溶剂如丙酮，从反胶束溶液中回收复性的核糖核酸酶A。这种沉淀方法对于从反胶束介质中回收蛋白质是有效的，且酶活性没有任何显著降低。
