Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2023;2705:307-348. doi: 10.1007/978-1-0716-3393-9_17.
The Src Homology 2 (SH2) domain is an emerging biotechnology with applications in basic science, drug discovery, and even diagnostics. The SH2 domains rapid uptake into different areas of research is a direct result of the wealth of information generated on its biochemical, biological, and biophysical role in mammalian cell biology. Functionally, the SH2 domain binds and recognizes specific phosphotyrosine (pTyr) residues in the cell to mediate protein-protein interactions (PPIs) that govern signal transduction networks. These signal transduction networks are responsible for relaying growth and stress state signals to the cell's nucleus, ultimately effecting a change in cell biology. Protein engineers have been able to increase the affinity of SH2 domains for pTyr while also tailoring the domains' specificity to unique amino acid sequences flanking the pTyr residue. In this way, it has been possible to develop unique SH2 variants for use in affinity-purification coupled to mass spectrometry (AP-MS) experiments, microscopy, or even synthetic biology. This chapter outlines methods to tailor the affinity and specificity of virtually any human SH2 domain using a combination of rational engineering and phage-display approaches.
Src 同源结构域 2（SH2）是一种新兴的生物技术，在基础科学、药物发现，甚至诊断领域都有应用。SH2 结构域迅速应用于研究的不同领域，这直接归因于其在哺乳动物细胞生物学中的生化、生物学和生物物理作用方面产生的丰富信息。从功能上讲，SH2 结构域结合并识别细胞中特定的磷酸酪氨酸（pTyr）残基，以介导蛋白质-蛋白质相互作用（PPIs），从而调控信号转导网络。这些信号转导网络负责将生长和应激状态信号传递到细胞的细胞核，最终影响细胞生物学的变化。蛋白质工程师能够提高 SH2 结构域与 pTyr 的亲和力，同时还可以根据 pTyr 残基侧翼的独特氨基酸序列对结构域的特异性进行定制。通过这种方式，已经有可能开发出用于亲和纯化与质谱（AP-MS）实验、显微镜，甚至合成生物学的独特 SH2 变体。本章概述了使用合理工程和噬菌体展示方法相结合来定制几乎任何人类 SH2 结构域的亲和力和特异性的方法。
