High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells.
High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells.
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哺乳动物细胞表达后膜蛋白的高通量生产和优化。
DOI：
10.1007/978-1-0716-3147-8_5
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Gamage N
中科院分区：
文献类型：
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作者：
Gamage N
高质量的蛋白质样品是任何结构生物学实验的基本要求。然而，生产高质量的蛋白质样品，尤其是膜蛋白样品，需要反复迭代且耗时。由于蛋白质产量低和高度不稳定性，尤其是当膜蛋白从其天然环境中去除时，膜蛋白结构生物学仍然具有挑战性。克服组成和构象不稳定的双重问题需要了解蛋白质大小、热稳定性和样品异质性，而并行方法可以同时分析多个条件。我们提出了一种方法，将膜蛋白构建体的高通量克隆与人胚胎肾（HEK）细胞中膜蛋白的瞬时表达相结合，并快速识别后续结构生物学应用的最合适条件。 Diamond Light Source 的膜蛋白实验室通常使用这种快速筛选方法来识别最成功的蛋白质构建体和条件，同时排除那些不起作用的蛋白质构建体和条件。 96 孔格式可轻松适应构建体、pH、盐、封装剂和其他添加剂（如脂质）的筛选。
High-quality protein samples are an essential requirement of any structural biology experiment. However, producing high-quality protein samples, especially for membrane proteins, is iterative and time-consuming. Membrane protein structural biology remains challenging due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Overcoming the twin problems of compositional and conformational instability requires an understanding of protein size, thermostability, and sample heterogeneity, while a parallelized approach enables multiple conditions to be analyzed simultaneously. We present a method that couples the high-throughput cloning of membrane protein constructs with the transient expression of membrane proteins in human embryonic kidney (HEK) cells and rapid identification of the most suitable conditions for subsequent structural biology applications. This rapid screening method is used routinely in the Membrane Protein Laboratory at Diamond Light Source to identify the most successful protein constructs and conditions while excluding those that will not work. The 96-well format is easily adaptable to enable the screening of constructs, pH, salts, encapsulation agents, and other additives such as lipids.