Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, United States.
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, United States.
Methods Enzymol. 2024;705:223-250. doi: 10.1016/bs.mie.2024.07.012. Epub 2024 Aug 15.
Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These> 200 kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.
酵母 Sen1 及其脊椎动物同源物 Senataxin（也称为 SETX）是 RNA-DNA 解旋酶。Sen1 和 SETX 参与多种关键的核功能，不仅限于但包括 DNA 复制和修复、RNA 加工和转录。这些 >200 kDa 的解旋酶具有双结构域架构，其 N 端调节性螺旋重复阵列通过大小可变的低复杂度序列的中央连接子与 SF1b 解旋酶马达核心连接。鉴于这些蛋白质的大小，生产适合生化、生物物理和蛋白质结构分析的毫克级蛋白质一直具有挑战性。为了克服这些限制，我们开发了一种稳健的可选择的高产 YFP 融合蛋白表达方法，用于哺乳动物细胞中 Sen1 的生产，然后通过高亲和力 YFP 结合骆驼纳米抗体支架进行纯化。在此，我们详细介绍了从嗜热真菌 Chaetomium thermophilum 表达和纯化重组 Sen1 的方法和方案，以及其 RNA-DNA 双链分辨率活性的定量表征。
