Ruan Biao, Fisher Kathryn E, Alexander Patrick A, Doroshko Viktoriya, Bryan Philip N
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Biochemistry. 2004 Nov 23;43(46):14539-46. doi: 10.1021/bi048177j.
Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.
枯草杆菌蛋白酶被改造成为一种高度特异性的加工蛋白酶，同时枯草杆菌蛋白酶原结构域也被共同改造成一个优化的识别序列。这涉及五个步骤。首先，构建了一个强大的枯草杆菌蛋白酶突变体，它能够耐受实现高特异性所需的后续突变。其次，对底物结合口袋进行突变以提高其序列选择性。第三，对枯草杆菌蛋白酶原结构域进行改造，使其能将切割作用导向与之融合的任何蛋白质的连接部位。第四，对枯草杆菌蛋白酶的活性位点进行改造，从动力学角度分离结合和切割反应。最后，鉴定出特定阴离子来触发加工反应，氟离子尤为有效。通过触发机制分离结合和加工步骤的能力创造了一种具有虚拟开关功能的蛋白酶。这使得固定在柱上的加工枯草杆菌蛋白酶既可用作亲和配体，又可用作加工蛋白酶，用于蛋白质的一步纯化。用改造后的原结构域标记的融合蛋白可与柱子结合，并洗去污染物。加入氟化物可触发切割反应，从而释放出纯的目标蛋白。然后通过在pH 2.1条件下洗脱紧密结合的原结构域来使柱子再生。迄今为止，已有十种蛋白质通过这种方法得到了纯化。
