Liu Bernard A, Ogiue-Ikeda Mari, Machida Kazuya
Broad Institute of MIT and Harvard, 415 Main St., 5175 JJ, Cambridge, MA, 02142, USA.
Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Ave., Farmington, CT, 06030, USA.
Methods Mol Biol. 2017;1555:117-162. doi: 10.1007/978-1-4939-6762-9_8.
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.
Src同源2（SH2）结构域是磷酸酪氨酸信号传导的核心，协调受体酪氨酸激酶（RTK）、接头蛋白和支架下游的信号传导事件。哺乳动物中存在一百多个SH2结构域，每个结构域都具有独特的特异性，决定了其与多个结合伴侣的相互作用。研究和确定SH2结构域在磷酸酪氨酸信号传导中作用所必需的重要工具之一是一组可溶性重组SH2蛋白。在此，我们基于对所有SH2结构域纯化的广泛经验，描述了用于蛋白质组学和基于生化研究（如肽阵列、质谱、蛋白质微阵列、反相微阵列和高通量荧光偏振（HTP-FP））所需的SH2结构域蛋白的生产方法。我们描述了使用GST或多聚组氨酸标签（两种广泛采用的亲和标签）表达和纯化SH2结构域的逐步方案。此外，我们讨论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SH2结构域的一般特征。
