Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Frankfurt/Main, Germany.
Methods Mol Biol. 2022;2507:405-424. doi: 10.1007/978-1-0716-2368-8_22.
Despite their importance in many essential physiological processes of living cells, G protein-coupled receptors (GPCRs) are often difficult to express and purify in sufficient quality and quantity. We demonstrate cell-free protein synthesis as an interesting alternative to classical cell-based expression systems. We focus on a recently developed detergent-free expression mode by co-translational integration of nascent GPCRs into provided nanodisc membranes of defined composition. The protocol is in particular suitable for detergent sensitive targets and allows the synthesis of full-length as well as modified GPCRs. As a basic blueprint for the cell-free synthesis of GPCRs and potentially other membrane proteins as well, we describe the production of the human endothelin-B receptor. Subsequent purification strategies are streamlined by implementing complementary affinity chromatography steps. We further show the evaluation and optimization of the final GPCR samples for homogeneity and activity through a radioligand binding assay.
尽管 G 蛋白偶联受体（GPCRs）在许多活细胞的重要生理过程中发挥着重要作用，但它们通常难以以足够的质量和数量进行表达和纯化。我们证明，无细胞蛋白质合成是一种替代经典基于细胞的表达系统的有趣方法。我们专注于最近开发的一种无去污剂表达模式，通过将新生 GPCR 共翻译整合到提供的具有明确定义组成的纳米盘膜中。该方案特别适用于对去污剂敏感的靶标，并允许全长和修饰的 GPCR 的合成。作为无细胞合成 GPCR 以及潜在其他膜蛋白的基本蓝图，我们描述了人类内皮素-B 受体的生产。通过实施互补亲和层析步骤，简化了随后的纯化策略。我们进一步通过放射性配体结合测定法展示了最终 GPCR 样品的均一性和活性的评估和优化。
