Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.
Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.
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DOI：
10.1016/s0065-3233(07)75003-9
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发表时间：
2008
影响因子：
--
通讯作者：
Joachimiak, Andrzej
中科院分区：
文献类型：
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作者：
Kim, Youngchang;Bigelow, Lance;Borovilos, Maria;Dementieva, Irina;Duggan, Erika;Eschenfeldt, William;Hatzos, Catherine;Joachimiak, Grazyna;Li, Hui;Maltseva, Natalia;Mulligan, Rory;Quartey, Pearl;Sather, Alicia;Stols, Lucy;Volkart, Lour;Wu, Ruiying;Zhou, Min;Joachimiak, Andrzej
在结构生物学中，最关键的问题是高质量样品的可用性。“结构生物学级”蛋白质必须以适合于使用X射线晶体学或核磁共振进行结构测定的数量和质量产生。结构基因组学的另一个挑战是需要低成本的大量蛋白质，其中蛋白质靶标通常具有低序列相似性，未知性质并且表征不佳。纯化程序必须可重复地产生含有毫克量标记原子的均质蛋白质或其衍生物。蛋白质纯化和处理程序的选择在获得高质量蛋白质样品中起着关键作用。结构生物学的最终目标是相同的-了解蛋白质在细胞过程中的结构基础，结构基因组学方法的不同之处在于，单个蛋白质或家族的功能方面并没有被忽视，但是，这里强调的是独特结构的数量，覆盖了大部分蛋白质折叠空间，并开发了高效率的新技术。在中西部中心结构基因组学（MCSG），我们已经开发了高通量平行蛋白质纯化的半自动化方案。简而言之，表达为与可切割亲和标签的融合物的蛋白质在两个固定化金属亲和层析（IMAC）步骤中纯化：（i）第一IMAC与缓冲液交换步骤偶联，并且在使用TEV蛋白酶切割标签后，（ii）第二IMAC和缓冲液交换以清除切割的标签和标记的TEV蛋白酶。也可根据需要应用尺寸排阻色谱法。这些方案已在多维色谱工作站AKTAexplorer和AKTAxpress（GE Healthcare）上实施。本章讨论了用于纯化的所有方法和方案，其中一些是在MCSG中开发的，另一些是采用并整合到MCSG纯化管道中的，最近是传染病结构基因组学中心（CSGID）纯化管道中的。
In structural biology, the most critical issue is the availability of high-quality samples. “Structural-biology-grade” proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same—to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Mid-west Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.