Levin Roman, Koeck Zoe, Dötsch Volker, Bernhard Frank
Institute of Biophysical Chemistry, Goethe University Frankfurt.
Institute of Biophysical Chemistry, Goethe University Frankfurt;
J Vis Exp. 2020 Nov 19(165). doi: 10.3791/61844.
Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.
无细胞表达系统允许对反应环境进行定制设计，以支持甚至像膜蛋白这样的复杂蛋白质的功能折叠。展示了将膜蛋白共翻译插入并折叠到作为纳米盘提供的预制且明确的膜中的实验程序。该方案完全不含去污剂，并且可以在一天内生成毫克级的纯化样品。所得的膜蛋白/纳米盘样品可用于各种功能研究和结构应用，如结晶、核磁共振或电子显微镜。描述了基本关键组件的制备，如无细胞裂解物、具有设计膜的纳米盘、关键储备溶液以及两室无细胞表达反应的组装。由于膜蛋白的折叠要求可能高度不同，该方案的一个主要重点是调节对样品质量重要的参数和反应步骤，如关键的基本反应化合物、纳米盘的膜组成、氧化还原和伴侣环境或DNA模板设计。整个过程通过视紫红质和G蛋白偶联受体的合成进行了演示。
