表2)。将溶于8 M urea中的scFv融合蛋白加入柱子顶端时，柱子的顶端也是8 M 的urea。而随着层析过程的进行，因为蛋白质的分子量远远大于小分子变性剂，被凝胶介质排阻在外，因此蛋白质的向下的移动速度要远大于各种小分子。这样scFv 融合蛋白就可以通过预先形成的urea 及pH梯度，并最后转移到复性缓冲液中(在柱子出口端占0.4 柱体积)。

表4. 流速对IMAC复性的影响

     通过逐步增加pH及降低urea的浓度，变性蛋白质可以在合适的氧化还原系统中逐步地复性成准确的天然结构。较低的起始pH可以使蛋白质在形成二硫键之前先折叠成一定的类似天然的空间结构，这样可以抑制错配的二硫键。流速也是优化复性过程的一个关键因素。如图4 所示，流速越大，复性率越低。当流速为1 ml/min 或更高时，蛋白质可以在2 小时内流到复性缓冲液中。时间较短，可以看作类似于稀释复性，而在此流速下的复性率也与稀释复性类似(表2，4)。在一定范围内，样品的体积对复性率的影响很小。增加GSSG与GSH并没有使复性率有明显提高，而且在此方法中精氨酸(arginine) 的浓度变化对复性率没有影响，
     因为urea本身也可以做为蛋白质复性过程中的聚集抑制剂。

     表2 中各种方法的比较说明脲及pH联合梯度对scFv57P 融合蛋白的复性有促进作用。只用脲梯度的复性率与IMAC的复性率相似。新方法促进融合蛋白复性的原因可能是因为将蛋白复性的过程分为两步：先在较低pH下通过逐步脱除脲抑制复性过程的聚集使蛋白质尽可能地折叠成类似天然的结构，然后通过逐步增加pH形成S-S 键。这样在类似天然的结构的基础上可以进一步的抑制错配S-S键(分子内或分子间) 的形成，从而达到进一步促进正确折叠的目的。

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