Gao Yong-Gui, Guan Yi-Xin, Yao Shan-Jing, Cho Man-Gi
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
Biotechnol Prog. 2003 May-Jun;19(3):915-20. doi: 10.1021/bp025775l.
The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.
伴侣蛋白mini-GroEL是一种可溶性重组片段，包含带有6xHis标签的GroEL的191-345氨基酸序列。研究了在mini-GroEL辅助下的复性方案对重组人干扰素γ包涵体活性恢复的影响。在悬浮体系中，mini-GroEL显著提高了重组人干扰素γ的活性恢复，在25℃下，mini-GroEL与重组人干扰素γ的化学计量比为1-5:1时效果最佳。此外，复性缓冲液中的1 M尿素对抑制聚集和提高活性恢复具有协同作用。最后，设计了一种新型色谱柱，柱顶含有1 cm高的Sephadex G 200，并填充固定化的mini-GroEL以促进复性。使用固定化mini-GroEL柱时，每毫克变性重组人干扰素γ回收的总活性高达3.93×10(6) IU，该柱可重复使用四次，复性能力无明显损失。开发了一种将伴侣蛋白制备和重组人干扰素γ体外折叠整合在一起的便捷技术。
