Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL, USA.
Adv Protein Chem Struct Biol. 2008;75:85-105. doi: 10.1016/S0065-3233(07)75003-9. Epub 2009 Feb 26.
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 Midwest 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.
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 X 射线晶体学或核磁共振结构测定的数量和质量产生。结构基因组学的额外挑战是需要以低成本获得大量蛋白质，其中蛋白质靶标往往具有低序列相似性、未知特性和较差的特征。纯化程序必须可重复地以毫克量产生均相蛋白质或其含有标记原子的衍生物。蛋白质纯化和处理程序的选择在获得高质量蛋白质样品方面起着关键作用。尽管结构生物学的最终目标是相同的 - 了解细胞过程中蛋白质的结构基础，但结构基因组学方法不同，因为单个蛋白质或家族的功能方面并没有被忽视，然而，这里的重点是独特结构的数量，涵盖了大部分蛋白质折叠空间，并开发了具有高效率的新技术。在中西部中心结构基因组学（MCSG），我们已经开发了用于高通量并行蛋白质纯化的半自动方案。简而言之，将蛋白质表达为与可切割亲和标签的融合物，通过两步固定化金属亲和层析（IMAC）进行纯化：（i）首先是与缓冲液交换步骤偶联的 IMAC，然后使用 TEV 蛋白酶切割标签，（ii）第二次 IMAC 和缓冲液交换，以清理切割标签和标记的 TEV 蛋白酶。如有需要，还可应用尺寸排阻层析。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 开发的，另一些是被采用并整合到 MCSG 纯化管道中，最近又被整合到传染病结构基因组学中心（CSGID）的纯化管道中。
