title: Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain.
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.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1.
authors.forename: Haiming
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada, N6A 5C1.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kaneko
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1. sachdev.sidhu@utoronto.ca.|Department of Molecular Genetics, University of Toronto, Donnelly Centre, 160 College Street, Toronto, ON, Canada, M5S 3E1. sachdev.sidhu@utoronto.ca.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: Department of Biochemistry and Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada, N6A 5C1. sli@uwo.ca.
authors.forename: Shawn S C
authors.initials: SS
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D019295:Computational Biology / Q000379:methods; D017124:Conserved Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D019143:Evolution, Molecular; D005822:Genetic Vectors / Q000235:genetics; D007698:Kinesis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software; D057188:Workflow; D018909:src Homology Domains / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D019151:Peptide Library; D019000:Phosphotyrosine
keywords: Directed evolution; Modular domain; Phage display; Phosphotyrosine; Post-translational modification; Src homology 2 (SH2) domain