Abstract:
In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.
摘要:
近年来,在蛋白质纯化和质量控制(QC)方面,新技术和策略的发展迅速,但是这些过程的基本技术可以追溯到很久以前,在过去的几十年里有很多改进。本章的目的是回顾这些方法,以及其他一些主题,例如细胞内或细胞外蛋白质的各种纯化方法的优缺点,最有效和广泛使用的基因工程亲和标签,溶解度增强标签,和用于去除非靶序列的特异性蛋白酶。亲和层析(AC),如用于回收抗体或Fc融合蛋白的蛋白A或G树脂或用于回收组氨酸标记蛋白的固定化金属,将与其他常规色谱技术一起讨论:离子交换(IEC),疏水交换(HEC),混合模式(MMC),尺寸排除(SEC),和超滤(UF)系统。如何选择和结合这些不同的技术来纯化任何给定的蛋白质,以及对纯度进行QC表征的最低标准,同质性,身份,和完整性的最终产品将呈现。