Chapter title |
Expression and Production of SH2 Domain Proteins
|
---|---|
Chapter number | 8 |
Book title |
SH2 Domains
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6762-9_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6760-5, 978-1-4939-6762-9
|
Authors |
Bernard A. Liu, Mari Ogiue-Ikeda, Kazuya Machida |
Editors |
Kazuya Machida, Bernard A. Liu |
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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