Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Frankfurt/Main, Germany.
Methods Mol Biol. 2021;2268:97-117. doi: 10.1007/978-1-0716-1221-7_7.
Cell-free protein expression systems and lipid nanoparticle technologies are core platforms for membrane protein synthesis. The implementation of preassembled nanodiscs allows the co-translational insertion of membrane proteins into tailored lipid bilayers in the absence of any artificial hydrophobic compounds. This strategy is particularly interesting for detergent sensitive or otherwise critical membrane proteins such as G-protein-coupled receptors (GPCRs). Cell-free expression reactions are completed within a day and the formed GPCR/nanodisc particles can be purified directly out of the reaction mixture by affinity tags and without any further manipulation. The streamlined procedure reduces risk of GPCR denaturation and the sample quality can further be supported by supplying chaperones or other beneficial compounds directly into the expression reactions.GPCRs inserted into nanoparticle membranes are excellent tools for a variety of applications such as ligand screening, engineering or even structural characterization. In this chapter, we provide protocols for the reaction set-up and efficient cell-free production of functionally folded GPCRs reaching μM concentrations in the final expression reactions. We further exemplify the tuning of GPCR sample quality and discuss their application for throughput ligand screening and for the analysis of ligand-binding characteristics.
无细胞蛋白表达系统和脂质纳米颗粒技术是膜蛋白合成的核心平台。预组装纳米盘的实施允许膜蛋白在没有任何人工疏水性化合物的情况下共翻译插入定制的脂质双层中。对于去污剂敏感或其他关键的膜蛋白（如 G 蛋白偶联受体（GPCR）），这种策略特别有趣。无细胞表达反应在一天内完成，形成的 GPCR/纳米盘颗粒可以通过亲和标签直接从反应混合物中纯化出来，而无需进行任何进一步的操作。简化的程序降低了 GPCR 变性的风险，并且可以通过直接向表达反应中提供伴侣蛋白或其他有益化合物来进一步支持样品质量。插入到纳米颗粒膜中的 GPCR 是各种应用的极好工具，例如配体筛选、工程甚至结构表征。在本章中，我们提供了反应设置的方案和高效的无细胞生产功能折叠 GPCR 的方案，最终表达反应中达到 μM 浓度。我们进一步说明了 GPCR 样品质量的调整，并讨论了它们在高通量配体筛选和配体结合特性分析中的应用。
