Journal article

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

Winnie Tan, Vincent J Murphy, Aude Charron, Sylvie van Twest, Michael Sharp, Angelos Constantinou, Michael W Parker, Wayne Crismani, Rohan Bythell-Douglas, Andrew J Deans

PLoS One | PUBLIC LIBRARY SCIENCE | Published : 2020

Abstract

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates o..

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Grants

Awarded by National Health and Medical Research Council, Australia


Awarded by Maddie Riewoldt's Vision


Awarded by NHMRC


Awarded by Maddie Riewoldt's Vision Fellow


Funding Acknowledgements

This work was supported by a grant from the National Health and Medical Research Council, Australia (GNT1123100 to A.J.D. and GNT1156343 to WC), Maddie Riewoldt's Vision (SVIMRV2017G) and the Victoria government IOS program. WT is recipient of a research training scholarship from the University of Melbourne, R. B-D is funded by the National Breast Cancer Foundation, W.C is an NHMRC Career Development Fellow (GNT1129757) and Maddie Riewoldt's Vision Fellow (WC-MRV2016) and AJD is a Victorian Cancer Agency mid-career fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.