Piserchio Andrea, Cowburn David, Ghose Ranajeet
Department of Chemistry, The City College of New York, New York, NY, USA.
Methods Mol Biol. 2012;831:111-31. doi: 10.1007/978-1-61779-480-3_7.
NMR analyses of the structure, dynamics, and interactions of the Src family kinases (SFKs) have been hindered by the limited ability to obtain sufficient amounts of properly folded, soluble protein from bacterial expression systems, to allow these studies to be performed in an economically viable manner. In this chapter, we detail our attempts to overcome these difficulties using the catalytic domain (SrcCD) of c-Src, the prototypical SFK, as an illustrative example. We describe in detail two general methods to express and purify SrcCD from Escherichia coli expression systems in both fully active wild-type and kinase-deficient mutant forms, allowing the efficient and cost-effective labeling by NMR-active isotopes for solution NMR studies.
从细菌表达系统中获取足够量的正确折叠的可溶性蛋白存在一定困难，这限制了对Src家族激酶（SFKs）的结构、动力学和相互作用进行核磁共振（NMR）分析，而以经济可行的方式进行这些研究也因此受到阻碍。在本章中，我们详细介绍了以典型的SFK——c-Src的催化结构域（SrcCD）为例，克服这些困难的尝试。我们详细描述了两种从大肠杆菌表达系统中表达和纯化SrcCD的通用方法，该蛋白可呈现完全活性的野生型和激酶缺陷型突变体形式，从而能够通过NMR活性同位素进行高效且经济高效的标记，用于溶液NMR研究。
