通过 MST 分析与 GaMD 模拟耦联的方法，实现核糖核蛋白复合物的高效纯化和组装，用于相互作用分析。
Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.
机构信息
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Center for Computational Biology and Department of Molecular Bioscience, University of Kansas, Lawrence, KS 66047, USA.
出版信息
STAR Protoc. 2021 Feb 3;2(1):100315. doi: 10.1016/j.xpro.2021.100315. eCollection 2021 Mar 19.
Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).
在这里，我们描述了一种使用重组 RNA 结合蛋白的可切割 GFP 融合蛋白监测蛋白质-RNA 相互作用的通用方案。我们详细介绍了每个表达和纯化步骤，包括高盐和肝素柱去除污染物 RNA。在 RNA 组装到核糖核蛋白复合物之后，使用微量热泳动分析（MicroScale Thermophoresis assay）可以快速获得结合亲和力，且仅需少量样品。进一步的高斯加速分子动力学模拟使我们能够详细分析蛋白质：RNA 相互作用。有关此方案使用和执行的完整详细信息，请参见 Gao 等人（2020 年）。
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