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Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations
Gao, Yunrong Cao, Dongdong Pawnikar, Shristi Akhter, Sana Miao, Yinglong Liang, Bo
Gao, Yunrong
Cao, Dongdong
Pawnikar, Shristi
Akhter, Sana
Miao, Yinglong
Liang, Bo
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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.
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Date
2021-03-19
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Publisher
Cell Press
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Keywords
Bioinformatics, Protein biochemistry, Protein expression and purification
Citation
Gao, Y., Cao, D., Pawnikar, S., Akhter, S., Miao, Y., & Liang, B. (2021). Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations. STAR protocols, 2(1), 100315. https://doi.org/10.1016/j.xpro.2021.100315