Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1.

Ho Yee Joyce Fung, Yuh Min Chook

Methods in molecular biology (Clifton, N.J.) 2022

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CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities. © 2022. Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Ho Yee Joyce Fung, Yuh Min Chook. Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1. Methods in molecular biology (Clifton, N.J.). 2022;2502:245-256