Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland.
Methods Mol Biol. 2022;2507:273-294. doi: 10.1007/978-1-0716-2368-8_14.
Structural studies of membrane proteins require high-quality samples. The target proteins should not only be pure and homogeneous but should also be active and allow the capture of a functionally relevant state. Here we present optimized methods for the expression and purification of human ABC transporters and oligosaccharyltransferase (OST) complexes that can be used for high-resolution structure determination using single-particle cryo-electron microscopy (cryo-EM). The protocols are based on the generation of stable cell lines that enable tetracycline-inducible expression of the target proteins. For the multidrug exporter ABCB1, we describe a protocol for reconstitution into nanodiscs and evaluation of the ATPase activity in the presence of drugs. For human OST, we describe a strategy for the purification of OST-A and OST-B complexes, including techniques to evaluate their integrity and activity using in vitro glycosylation assays. These protocols can be adapted for the production of other human ABC transporters and multimeric membrane protein complexes.
膜蛋白的结构研究需要高质量的样品。目标蛋白不仅应纯净且均一，还应具有活性，并能捕获具有相关功能的状态。在这里，我们介绍了优化的方法，用于表达和纯化人 ABC 转运蛋白和寡糖基转移酶 (OST) 复合物，这些方法可用于使用单颗粒冷冻电子显微镜 (cryo-EM) 进行高分辨率结构测定。这些方案基于生成稳定的细胞系，使目标蛋白能够进行四环素诱导表达。对于多药外排泵 ABCB1，我们描述了一种将其重构成纳米盘的方案，并描述了在存在药物的情况下评估其 ATP 酶活性的方法。对于人 OST，我们描述了一种纯化 OST-A 和 OST-B 复合物的策略，包括使用体外糖基化测定评估其完整性和活性的技术。这些方案可以适应于生产其他人类 ABC 转运蛋白和多聚体膜蛋白复合物。
