Developing a toolbox of Affimer reagents targeting SH2 domains to study protein-protein interactions in disease

Src Homology 2 (SH2) domains are phosphotyrosine-binding modules that mediate a range of protein-protein interactions. These domains are found in over 120 human proteins and are involved in several signalling pathways that can become deregulated in diseases such as cancer. Research into the role of individual SH2s in disease has been hampered by a lack of protein-specific reagents available for intracellular functional assays. Specificity of reagents is difficult to achieve, due to the high sequence and structural homology of the domains. Research presented in this thesis investigates the use of a novel non-immunoglobulin binding protein, the Affimer, as an SH2 domain research reagent. The project aimed to isolate Affimer reagents that bound to SH2 domains in a protein-specific manner, and to explore their ability to inhibit their target in several in vitro assays. Affimer binders were raised against 38 SH2 domains and tested in protein microarrays to determine specificity; revealing the isolation of 62 protein-specific reagents. A subset of Grb2 SH2-binding Affimer reagents were used in in vitro characterisation studies, demonstrating high binding affinities for their target and competitive inhibition of the domain. Selected binders used in mammalian cell-based assays also showed disruption of Grb2-mediated signalling, evidenced by reduced phosphorylation of a downstream target. The SH2-binding Affimer reagents tested in this study showed comparable qualities to other previously SH2-targeting binding proteins, with many displaying higher specificity for their target. The Affimer therefore has good potential for use in the study of SH2 domain-mediated signalling. In particular, utilising SH2-binding Affimer proteins as a screening tool in functional cell-based assays is an exciting future prospect.