School of Food Science and Environmental Health, Technological University Dublin, Grangegorman, Dublin, Ireland.
Methods Mol Biol. 2023;2699:77-95. doi: 10.1007/978-1-0716-3362-5_6.
All cells contain proteases, which hydrolyze the peptide bonds between amino acids of a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed.
所有细胞都含有蛋白酶，这些酶可以水解蛋白质主链氨基酸之间的肽键。通常，蛋白酶通过调节和物理分离到不同的细胞区室来防止非特异性蛋白水解；然而，在细胞裂解过程中，这种分离并不保留，细胞裂解是任何蛋白质分离程序的初始步骤。在蛋白质纯化过程中防止蛋白水解通常采用双管齐下的方法：首先，原位抑制蛋白水解，然后通过色谱纯化将蛋白酶与感兴趣的蛋白质早期分离。蛋白酶抑制剂通常用于在通过柱色谱从感兴趣的蛋白质中物理分离之前限制蛋白酶的作用。本章综述了在蛋白质表达和纯化过程中减少或避免蛋白水解的常用方法。
