Expression and Purification of Microtubule-Associated Proteins from HEK293T Cells for In Vitro Reconstitution
Expression and Purification of Microtubule-Associated Proteins from HEK293T Cells for In Vitro Reconstitution
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HEK293T 细胞微管相关蛋白的表达和纯化用于体外重建
DOI：
10.1007/978-1-0716-0219-5_2
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发表时间：
2020-01-01
期刊：
影响因子：
--
通讯作者：
Jiang, Kai
中科院分区：
文献类型：
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作者：
Hua, Shasha;Jiang, Kai
体外重组已成为阐明微管相关蛋白（MAPs）的作用和机制的宝贵工具。与所有生化分析一样，蛋白质的质量对于成功至关重要。在微管领域，细菌和昆虫细胞中产生的蛋白质已被广泛用于体外重构。最近，我们将哺乳动物HEK293T细胞表达系统应用于我们对几种MAP的研究。我们发现这种系统特别适合于快速功能研究，并且可以产生细菌或昆虫细胞表达系统有时难以产生的活性蛋白。在这里，我们提供了一个详细的协议，表达和纯化微管相关蛋白从HEK293T细胞使用Strep标签策略。本文所述的方法可用于制备用于重构研究的其他蛋白质和蛋白质复合物。
In vitro reconstitution has been an invaluable tool to elucidate the roles and mechanisms of microtubule-associated proteins (MAPs). Like all biochemical assays, the quality of the proteins is vital for success. In the microtubule field, proteins produced in bacteria and insect cells have been widely used for in vitro reconstitution. Recently, we applied the mammalian HEK293T cell expression system to our research on several MAPs. We find that such system is especially suitable for quick functional studies and can produce active proteins that sometimes are difficult for either bacteria or insect cell expression systems. Here, we provide a detailed protocol to express and purify microtubule-associated proteins from HEK293T cells using a Strep-tag strategy. The method described here can be adopted for preparation of other proteins and protein complexes for reconstitution studies.