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

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Book cover The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

Abstract

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.

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Acknowledgments

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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Authors and Affiliations

  1. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada

    Wei Zhang & Sachdev S. Sidhu

  2. Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada

    Sachdev S. Sidhu

  3. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

    Sachdev S. Sidhu

Authors
  1. Wei Zhang
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  2. Sachdev S. Sidhu
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Corresponding authors

Correspondence to Wei Zhang or Sachdev S. Sidhu .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

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Zhang, W., Sidhu, S.S. (2018). Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_8

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8705-4

  • Online ISBN: 978-1-4939-8706-1

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