Ogiue-Ikeda Mari, Machida Kazuya
Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06030, USA.
Methods Mol Biol. 2017;1555:199-223. doi: 10.1007/978-1-4939-6762-9_12.
Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies.
重组模块化蛋白结构域一直是用于解读蛋白质-蛋白质相互作用以及阐明蛋白质修饰在细胞信号传导中作用的便捷蛋白质组学工具。为了获得可靠的实验数据，这些蛋白质结构域探针需要足够的特异性和灵敏度。由于天然进化的蛋白质结构域并不总是具有用于体外分析的最佳生化特性，有时需要进行功能改变，如提高亲和力。在本章中，我们描述了功能缺失型和串联SH2结构域的制备方法，这些方法应广泛适用于高通量和低通量磷酸化蛋白质组学研究。
