Purification and Characterization of Proteins
Purification and Characterization of Proteins
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DOI：
10.1128/9781555816827.ch52
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
U. Strych;R. Willson
中科院分区：
文献类型：
--
作者：
U. Strych;R. Willson
本章介绍了蛋白质纯化和表征的迷人的，有用的，有时可怕的世界。纯化的目的随着纯化蛋白的预期用途而变化。蛋白质纯化通常可以分为五个主要阶段，这些阶段不需要按顺序进行：（i）源的制备，（ii）收集关于蛋白质性质的所有可用信息，（iii）开发测定法，（iv）初步分离，和（v）最终纯化。近年来，最初由Studier开发、现在由EMD Chemicals分销的自诱导培养基在蛋白质表达界获得了越来越多的追随者。回收和初始分离步骤将产物与培养基中的大部分水和大部分宿主细胞组分分离。因此，细胞外和细胞内蛋白质的回收通常始于通过离心或过滤将可溶性培养基与不溶性细胞沉淀分离。离子交换色谱法是制备分离蛋白质的最常见的高分辨率方法，并用于大多数方案中。许多真核生物（和少数原核生物）蛋白质含有糖，糖有时对蛋白质的功能至关重要。例如，我们现在知道糖基化蛋白质参与了各种重要的分子识别事件。可用的方法包括用于检测糖基化的染色和印迹、去糖基化酶、用于印迹和分离的糖特异性凝集素、以及表征单个侧链的核磁共振和质谱（MS）方法。
This chapter provides an introduction to the fascinating, useful, and sometimes dreaded world of protein purification and characterization. The goals of purification vary with the intended use of the purified protein. Protein purification can generally be divided into five broad stages, which do not all need to occur sequentially: (i) preparation of the source, (ii) gathering of all available information about the protein’s properties, (iii) development of an assay, (iv) primary isolation, and (v) final purification. In recent years an autoinduction medium originally developed by Studier and now distributed by EMD Chemicals has gained an increased following in the protein expression community. Recovery and initial isolation steps separate the product from the majority of the water in the cultivation medium and from the majority of the host cell components. Recovery of both extracellular and intracellular proteins thus usually begins with the separation of the soluble culture medium from the insoluble cell pellet by centrifugation or filtration. Ion-exchange chromatography is the most common high-resolution method for preparative separation of proteins and is used in most protocols. Many eukaryotic (and a few prokaryotic) proteins contain sugars, which are sometimes essential to the protein’s function. For instance, we now know that glycosylated proteins are involved in a wide variety of crucial molecular recognition events. Available methods include stains and blots for detection of glycosylation, deglycosylating enzymes, sugar-specific lectins for blotting and separations, and nuclear magnetic resonance and mass spectrometry (MS) methods of characterizing individual side chains.