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Expression and Production of SH2 Domain Proteins

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SH2 Domains

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

Abstract

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

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Acknowledgments

We thank Kin Leung, Richard B. Jones, Piers Nash, and Brett Engelmann for sharing their protocols on SH2 domains. We thank Joshua Jadwin for assistance with editing the manuscript, and Bruce Mayer for his continuous encouragement and support. This study was partly supported by grant CA1154966 from the National Institutes of Health and Quest for CURES (QFC) grant from the Leukemia and Lymphoma Society (to K.M.).

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Authors and Affiliations

  1. Broad Institute of MIT and Harvard, 415 Main St., 5175 JJ, Cambridge, MA, 02142, USA

    Bernard A. Liu

  2. Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Ave., Farmington, CT, 06030, USA

    Mari Ogiue-Ikeda & Kazuya Machida

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  1. Bernard A. Liu
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  2. Mari Ogiue-Ikeda
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  3. Kazuya Machida
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Correspondence to Kazuya Machida .

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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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Liu, B.A., Ogiue-Ikeda, M., Machida, K. (2017). Expression and Production of SH2 Domain Proteins. 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6762-9_8

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