[目的]建立抗人胰岛素样生长因子1受体单链抗体包涵体复性方法。[方法]首先,在96孔板上进行稀释复性,从72种复性液中筛选最佳条件。每孔复性液2 m L,滴入起始浓度1. 5 mg/m L的包涵体溶解液100μL过夜复性。然后,选择最佳复性液与变性液混合在Superdex 75柱上形成1 cm柱长下降5%的变性液梯度,样品按5%柱体积上样进行柱上复性。[结果]最适稀释复性液为C8(50 mmol/L Tris-Cl,GSH/GSSG=5/0. 5 mmol/L,0. 4 mol/L精氨酸,p H 9.0),对应复性率约为78%;以该复性液为基础通过柱上复性,目标蛋白复性率提高到95%,复性样品抗原结合活性良好。[结论]建立了目标蛋白包涵体柱上复性方法,复性率达到95%,产量达到384 mg/L。
【英文摘要】
[Objective] This study was to develop a renaturation method for the inclusion body of a recombinant Single-chain variable fragment( Sc Fv) against Insulin-like growth factor 1 receptor( IGF-1 R). [Methods] Firstly,a dilution refolding was performed in 96-well plate to identify the optimal condition from 72 kinds of refolding buffers. Each plate was pre-filled with 2 m L refolding buffer,and then added 100 μL denatured inclusion body solution,which was followed by an overnight incubation. Secondly,an on-colu...
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