Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries

  • Wei ZhangEmail author
  • Sachdev S. SidhuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)


Ubiquitination is a posttranslational protein modification pathway regulating diverse cellular processes that are implicated in numerous human diseases. However, targeting the enzymes in the ubiquitination cascade potently and selectively remains a major challenge. Recently we devised a methodology to generate ubiquitin-based modulators for E3 ligases and deubiquitinases, enzymes that control the specificity of protein ubiquitination and deubiquitination, respectively. Here, we describe methods to generate libraries of ubiquitin variants and perform phage display selections to isolate high-affinity binders for target proteins. Importantly, the strategy introduced here can be applied to other small protein domains mediating protein-protein interactions to engineer tools for target validation and potential therapeutic development.

Key words

Phage display Combinatorial library Ubiquitin Inhibitor Activator E3 ligase Deubiquitinase 



We acknowledge the technical assistance and work summary from Mr. Jun Gu. We greatly appreciate the help from past and present collaborators, including Drs. Andreas Ernst, Jason Moffat, Brenda A. Schulman, J. Wade Harper, Daniela Rotin, Danny T. Huang, Brian L Mark, and Marjolein Kikkert. This work is supported by the Canadian Institutes of Health Research (CIHR) project grant (#0000303157) awarded to S.S.S.


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Donnelly Centre for Cellular and Biomolecular ResearchUniversity of TorontoTorontoCanada
  2. 2.Banting and Best Department of Medical ResearchUniversity of TorontoTorontoCanada
  3. 3.Department of Molecular GeneticsUniversity of TorontoTorontoCanada

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