Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, UK.
The Rosalind Franklin Institute, Didcot, UK.
Methods Mol Biol. 2021;2305:105-128. doi: 10.1007/978-1-0716-1406-8_5.
Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.
癌症、神经退行性疾病和传染病仍然是全球主要死亡原因之一。结构导向的药物设计对于推进这些重要疾病的药物研发至关重要。结构测定工作流程中的一个关键挑战是生产高质量的真核膜蛋白（药物靶点）。已经开发了许多表达系统来生产真核膜蛋白。本章介绍了在 Expi293F 细胞中转染和表达真核膜蛋白的优化详细方案。小规模测试表达和纯化可以优化用于下游结构（冷冻电镜）解析的样品制备条件。
