Sun Weimin, Dai Xinxian, Zheng Yanpeng, Wang Jianwei, Hou Lingling, Du Juan, Hu Hong-gang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing 100044, China.
Protein Expr Purif. 2011 Jan;75(1):83-8. doi: 10.1016/j.pep.2010.09.009. Epub 2010 Sep 17.
Protein refolding is a bottleneck in the production of therapeutic proteins from inclusion bodies. In recent years, several studies have described on-column refolding of recombinant proteins. DT389-hIL13 is a recombinant protein that targets the glioma. In our study, the recombinant protein DT389-hIL13 was expressed in Escherichia coli (E. coli). The isolated inclusion bodies were refolded using size exclusion chromatography (SEC) and further purified using anion exchange chromatography. Three different methods of SEC on-column refolding were studied. In vitro tests on U251 cells showed that the recombinant protein could effectively inhibit the proliferation of U251 cells, especially the protein refolded by urea and pH gradient method. The half-maximal inhibitory concentration (IC50) of 0.887 nM was achieved with this new method, unlike an IC50 of 11.4 nM achieved in the non-gradient method.
蛋白质复性是从包涵体生产治疗性蛋白质过程中的一个瓶颈。近年来,有几项研究描述了重组蛋白的柱上复性。DT389-hIL13是一种靶向胶质瘤的重组蛋白。在我们的研究中,重组蛋白DT389-hIL13在大肠杆菌中表达。分离得到的包涵体使用尺寸排阻色谱(SEC)进行复性,并进一步使用阴离子交换色谱进行纯化。研究了三种不同的SEC柱上复性方法。对U251细胞的体外测试表明,重组蛋白能够有效抑制U251细胞的增殖,尤其是通过尿素和pH梯度法复性的蛋白。用这种新方法实现了0.887 nM的半数最大抑制浓度(IC50),而在非梯度法中IC50为11.4 nM。