title: Expression and Production of SH2 Domain Proteins.
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.
authors.affiliation: Broad Institute of MIT and Harvard, 415 Main St., 5175 JJ, Cambridge, MA, 02142, USA.
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Mari
authors.initials: M
authors.lastname: Ogiue-Ikeda
authors.affiliation: 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. machida@uchc.edu.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D018909:src Homology Domains
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins
keywords: Bacterial expression; GST tag; His tag; Protein purification; SH2 domains