Fan Xiangdong, Xu Diansheng, Lu Bing, Xia Jie, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
J Biochem Biophys Methods. 2008 Apr 24;70(6):1130-8. doi: 10.1016/j.jprot.2007.12.004. Epub 2008 Jan 7.
Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme.
包涵体复性过程在重组蛋白的生产中起着重要作用。本文通过将递减的尿素梯度与递增的精氨酸梯度（双梯度）相结合，实现了用于新型溶栓剂NTA蛋白复性的尺寸排阻色谱复性过程的改进。针对提高NTA蛋白活性回收率和抑制聚集，研究了双梯度凝胶过滤过程中的不同复性方法和不同操作条件。变性NTA蛋白的复性表明，该方法可在高蛋白浓度下显著提高蛋白的活性回收率。从初始NTA蛋白浓度至20 mg/ml时，活性回收率达到37%。本研究所得出的结论也可应用于溶菌酶的复性。
