Membrane Protein Production in Insect Cells.
Membrane Protein Production in Insect Cells.
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昆虫细胞中膜蛋白的产生。
DOI：
10.1007/978-1-0716-2368-8_12
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Vaitsopoulou A
中科院分区：
文献类型：
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作者：
Vaitsopoulou A
膜蛋白是生命机器的重要组成部分。它们连接细胞的内部和外部，在细胞信号传递中发挥重要作用，并负责营养物质和代谢物的流入和流出。为了分析它们的结构和功能，需要高产量的正确折叠和修饰的蛋白质。昆虫细胞，如Sf9细胞，在过去十年的结构研究中一直是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，比微生物系统提供更自然的翻译后修饰。在这里，我们描述了建立和维护昆虫细胞培养物的一般技术，使用FlashBac™或Bac-to-Bac™系统产生和扩增重组杆状病毒群，膜蛋白生产，以及用于提取和纯化实验的膜制剂的生产。
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 asSf9cells, 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.