High-throughput protein purification and quality assessment for crystallization.
High-throughput protein purification and quality assessment for crystallization.
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DOI：
10.1016/j.ymeth.2011.07.010
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发表时间：
2011-09
期刊：
影响因子：
4.8
通讯作者：
Joachimiak, Andrzej
中科院分区：
文献类型：
--
作者：
Kim, Youngchang;Babnigg, Gyorgy;Jedrzejczak, Robert;Eschenfeldt, William H.;Li, Hui;Maltseva, Natalia;Hatzos-Skintges, Catherine;Gu, Minyi;Makowska-Grzyska, Magdalena;Wu, Ruiying;An, Hao;Chhor, Gekleng;Joachimiak, Andrzej
关键词：
结构生物学的最终目标是了解蛋白质在细胞过程中的结构基础。在结构生物学中，最关键的问题是高质量样本的可用性。“结构生物级”蛋白质的产生数量和质量必须适合于使用X射线结晶学或核磁共振光谱学进行结构测定。纯化过程必须可重复地产生均一蛋白质或含有标记原子(S)的衍生物(毫克量)。蛋白质纯化和处理程序的选择对获得高质量的蛋白质样品起着至关重要的作用。随着结构基因组学强调以基因组为基础的方法来理解蛋白质的结构和功能，人们已经确定了一些覆盖大部分蛋白质折叠空间的独特结构，并开发了高效的新技术。在中西部结构基因组学中心(MCSG)，我们已经开发了高通量平行蛋白质表达和纯化的半自动方案。表达为与可切割亲和标签的融合的蛋白质在两个连续的固定化金属亲和层析(IMAC)步骤中被纯化：(I)第一步是与缓冲交换或尺寸排除层析(IMAC-I)耦合的IMAC，随后使用高度特异的烟草蚀刻病毒(TEV)蛋白酶切割亲和标签；第二步骤是IMAC和缓冲交换(IMAC-II)以去除切割的标签和标记的TEV蛋白酶。这些方案已经在多维层析工作站上实现，正如我们已经展示的那样，许多蛋白质可以成功地大规模生产。本章讨论了用于纯化的所有方法和方案，其中一些方法由MCSG开发，另一些采用并集成到MCSG纯化管道中，最近被整合到传染病结构基因组学中心(CSGID)的纯化管道中。
The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. 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. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed in this chapter.
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DOI：
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发表时间：
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期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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作者：
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通讯作者：
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期刊：
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影响因子：
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作者：
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DOI：
10.1107/s0907444906047020
发表时间：
2007-01
期刊：
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影响因子：
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作者：
Hassell AM;An G;Bledsoe RK;Bynum JM;Carter HL 3rd;Deng SJ;Gampe RT;Grisard TE;Madauss KP;Nolte RT;Rocque WJ;Wang L;Weaver KL;Williams SP;Wisely GB;Xu R;Shewchuk LM
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Shewchuk LM