Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.

• Type of Material: Article
• Authors: Yunrong Gao, Emory University; Dongdong Cao, Emory University; Shristi Pawnikar, University of Kansas; Sana Akhter, University of Kansas; Yinglong Miao, University of Kansas; Bo Liang, Emory University
• Language: English
• Date: 2021-03-19
• Publisher: Cell Press
• Publication Version: Final Published Version
• Copyright Statement: © 2021 The Author(s)
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.xpro.2021.100315
• Title of Journal or Parent Work: STAR Protoc
• Volume: 2
• Issue: 1
• Start Page: 100315
• End Page: 100315
• Grant/Funding Information: B.L. was supported by NIGMS R01GM130950 and the Start-Up Fund from Emory University School of Medicine. Y.M. was supported by startup funding from the College of Liberal Arts and Sciences at the University of Kansas.
• Keywords: Protein biochemistry; Bioinformatics; Protein expression and purification
• Research Categories: Biology, Bioinformatics; Biology, Cell

Abstract

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).