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Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain
Haiming Huang1,2, Tomonori Kaneko3, Sachdev S Sidhu4,5
1Banting and Best Department of Medical Research, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1.
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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