Expression and Production of SH2 Domain Proteins.
Expression and Production of SH2 Domain Proteins.
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DOI：
10.1007/978-1-4939-6762-9_8
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发表时间：
2017
影响因子：
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通讯作者：
Bernard A. Liu;M. Ogiue‐Ikeda;K. Machida
中科院分区：
文献类型：
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作者：
Bernard A. Liu;M. Ogiue‐Ikeda;K. Machida
Src同源2(SH2)结构域位于磷酸化酪氨酸信号的核心，协调受体酪氨酸激酶(RTK)、接头和支架下游的信号事件。哺乳动物中存在超过100个SH2结构域，每个结构域都有独特的特异性，决定了它与多个结合伙伴的相互作用。研究和确定SH2结构域在磷酸酪氨酸信号转导中的作用所必需的工具之一是一组可溶的重组SH2蛋白。在这里，我们描述了基于纯化所有SH2结构域的广泛经验，生产蛋白质组学和基于生物化学研究所需的SH2结构域蛋白的方法，如肽阵列、质谱学、蛋白质微阵列、反相微阵列和高通量荧光偏振(HTP-FP)。我们描述了使用GST或PolyHis-tag这两种广泛采用的亲和标签来表达和纯化SH2结构域的分步方案。此外，我们还介绍了评估蛋白质质量的替代方法、挑战和验证研究，并提供了纯化的人类SH2结构域的一般特征。
The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.