College of Health and Life Sciences, Aston University, Birmingham, UK.
Methods Mol Biol. 2022;2507:223-240. doi: 10.1007/978-1-0716-2368-8_12.
Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.
膜蛋白是生命机器的重要组成部分。它们连接着细胞的内外，在细胞信号转导中发挥着重要作用，负责营养物质和代谢物的流入和流出。为了对其进行结构和功能分析，需要获得大量正确折叠和修饰的蛋白质。在过去的十年中，昆虫细胞（如 Sf9 细胞）已成为结构研究中真核膜蛋白的主要表达宿主之一，因为它们比哺乳动物细胞更容易处理，并提供比微生物系统更自然的翻译后修饰。在这里，我们描述了建立和维持昆虫细胞培养物、使用 flashBAC™ 或 Bac-to-Bac™ 系统生成和扩增重组杆状病毒株、膜蛋白生产以及为提取和纯化实验生产膜制剂的一般技术。
