Abdine Alaa, Park Kyu-Ho, Warschawski Dror E
CNRS and Université Paris Diderot, IBPC, Paris, France.
Methods Mol Biol. 2012;831:85-109. doi: 10.1007/978-1-61779-480-3_6.
Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.
尽管无细胞表达在生化工具库中算是相对较新的技术，但它已经得到了广泛的综述，甚至在诸如膜蛋白表达、纳米脂蛋白颗粒以及在晶体学和核磁共振（NMR）中的应用等更专业的领域也是如此。固态NMR在结构生物学工具库中同样是一项新技术，在样品制备方面有其自身的特点。无细胞表达和固态NMR是一种很有前景的组合，已被证明对研究天然环境（即水合脂质双层）中的膜蛋白结构很有用。我们在下面描述了几种使用无细胞表达制备机械敏感膜通道MscL的方案，这些方案旨在用于固态NMR研究。这些方案具有灵活性，稍作调整即可轻松应用于其他膜蛋白。
