Rues Ralf-Bernhardt, Gräwe Alexander, Henrich Erik, Bernhard Frank
Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, 60438, Frankfurt/Main, Germany.
Methods Mol Biol. 2017;1586:291-312. doi: 10.1007/978-1-4939-6887-9_19.
Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.
无细胞表达能够以全新的形式合成膜蛋白，这些形式相对容易针对特定应用进行定制。两亲性超结构，如胶束、脂质体或纳米盘，可以作为纳米装置用于膜蛋白的溶解。纳米盘形式的确定的空双层为膜蛋白提供了类似天然的环境，支持功能折叠、正确的寡聚组装以及稳定性。通过将纳米盘技术与高效的无细胞表达系统相结合，即使是非常难处理且对去污剂敏感的膜蛋白也能得到解决，因为新生膜蛋白有可能直接共翻译插入到提供的预组装纳米盘中。本章提供了在预组装纳米盘存在下合成膜蛋白的更新方案，适用于脂质对膜蛋白功能影响的筛选和寡聚复合物形成的调控等新兴应用。
