title: Functionally Altered SH2 Domains for Biochemical Studies: Loss-of-Function Mutant and Domain Concatenation.
abstract: Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies.
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 Avenue, 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 Avenue, Farmington, CT, 06030, USA. machida@uchc.edu.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D000073658:Loss of Function Mutation; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D020080:Tandem Repeat Sequences; D018909:src Homology Domains / Q000235:genetics
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins
keywords: Domain concatenation; Loss of function; SH2 domain; Tandem SH2