Saijo-Hamano Y, Namba K, Oosawa K
Protonic NanoMachine Project, ERATO, JST, 1-7 Hikaridai, Seika 619-0237, Japan.
J Struct Biol. 2000 Nov;132(2):142-6. doi: 10.1006/jsbi.2000.4311.
Proteolysisis a major problem in purification of overproduced proteins for structural studies. We developed a new method to avoid proteolysis of the products even in cases where popular protease inhibitors do not work effectively. When we cloned FlgF, a flagellar rod protein, from Salmonella typhimurium and overproduced it in Escherichia coli, FlgF was highly susceptible to cleavage by endogenous proteases after cell disruption even in the presence of various protease inhibitors. However, FlgF was not digested when the cells were disrupted in the presence of urea, which allowed us to develop the following new purification procedure. After cell disruption in the presence of urea and removal of the cell debris, the supernatant was passed through tandem-connected cation- and anion-exchange columns. Proteases were trapped in the cation-exchange column, and protease-free FlgF was eluted from the disconnected anion-exchange column. This gave a stable full-length product suitable for crystallization trials. The key procedures are cell disruption in the presence of urea and linked ion-exchange chromatography to quickly remove proteases as well as urea. This fast and simple method can be applied to purification of other overproduced proteins that are very sensitive to proteolysis.
在为结构研究而纯化过量表达的蛋白质时，蛋白水解是一个主要问题。我们开发了一种新方法，即使在常用蛋白酶抑制剂效果不佳的情况下，也能避免产物发生蛋白水解。当我们从鼠伤寒沙门氏菌中克隆鞭毛杆蛋白FlgF并在大肠杆菌中过量表达时，即使存在各种蛋白酶抑制剂，细胞破碎后FlgF仍极易被内源性蛋白酶切割。然而，当在尿素存在的情况下破碎细胞时，FlgF未被消化，这使我们得以开发以下新的纯化程序。在尿素存在下破碎细胞并去除细胞碎片后，将上清液通过串联的阳离子和阴离子交换柱。蛋白酶被困在阳离子交换柱中，无蛋白酶的FlgF从断开连接的阴离子交换柱上洗脱下来。这得到了适合进行结晶试验的稳定全长产物。关键步骤是在尿素存在下破碎细胞以及采用连接离子交换色谱法快速去除蛋白酶和尿素。这种快速且简单的方法可应用于纯化其他对蛋白水解非常敏感的过量表达的蛋白质。
