SARS-CoV-2 3a 蛋白的表达、纯化和脂质纳米盘重建。
SARS-CoV-2 3a expression, purification, and reconstitution into lipid nanodiscs.
机构信息
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States; Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, United States; California Institute for Quantitative Biology (QB3), University of California Berkeley, Berkeley, CA, United States.
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States; Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, United States; California Institute for Quantitative Biology (QB3), University of California Berkeley, Berkeley, CA, United States.
出版信息
Methods Enzymol. 2021;653:207-235. doi: 10.1016/bs.mie.2020.12.020. Epub 2021 Jan 27.
The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.
SARS-CoV-2 的 3a 蛋白是一种假定的离子通道，与病毒的生命周期和发病机制有关。我们最近表达、纯化并将 3a 重构成脂质纳米盘，通过低温电镜将其结构解析至 2.1Å 分辨率。在本章中，我们描述了为促进其他实验室对该蛋白进行研究而开发的方法。我们强调了使从基因序列到重组蛋白（3a 的情况下为 3 周）的快速进展的因素，并提供了一般观察结果和技巧，以将这些方案应用于其他感兴趣的膜蛋白。
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