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SH2 Domains pp 225–254Cite as

Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1555))

Abstract

Commercial antibodies raised against phosphotyrosine have been widely used as reagents to detect or isolate tyrosine-phosphorylated proteins from cellular samples. However, these antibodies are costly and are not amenable to in-house production in an academic lab setting. In this chapter, we describe a method to generate super-high affinity SH2 domains, dubbed the phosphotyrosine superbinders, by evolving a natural SH2 domain using the phage display technology. The superbinders are stable and can be easily produced in Escherichia coli in large quantities. The strategy presented here may also be applied to other protein domains to generate domain variants with markedly enhanced affinities for a specific post-translational modification.

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Acknowledgements

Work described here was supported, in part, by grants from the Canadian Cancer Society Research Institute and the Ontario Research Fund. SSCL holds a Canada Research Chair in Functional Genomics and Cellular Proteomics.

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Author notes

    Authors and Affiliations

    1. Banting and Best Department of Medical Research, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1

      Haiming Huang & Sachdev S. Sidhu

    2. Department of Molecular Genetics, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1

      Haiming Huang & Sachdev S. Sidhu

    3. Department of Biochemistry and Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada, N6A 5C1

      Tomonori Kaneko & Shawn S. C. Li

    Authors
    1. Haiming Huang
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    2. Tomonori Kaneko
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    3. Sachdev S. Sidhu
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    4. Shawn S. C. Li
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    Corresponding authors

    Correspondence to Sachdev S. Sidhu or Shawn S. C. Li .

    Editor information

    Editors and Affiliations

    1. Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, Connecticut, USA

      Kazuya Machida

    2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

      Bernard A. Liu

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    Huang, H., Kaneko, T., Sidhu, S.S., Li, S.S.C. (2017). Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain. In: Machida, K., Liu, B. (eds) SH2 Domains. Methods in Molecular Biology, vol 1555. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6762-9_13

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    • DOI: https://doi.org/10.1007/978-1-4939-6762-9_13

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6760-5

    • Online ISBN: 978-1-4939-6762-9

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