(1297-B) Generating affinity reagents to phosphoepitopes by phage-display
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Antibodies have played a particularly useful role in monitoring post-translational modifications, such as phosphorylated threonine resides, which often regulate a protein's biochemical activity and cellular location. While the vast majority of commercial antibodies to phosphoepitopes are polyclonal and monoclonal antibodies, they are limited in renewability and protein engineering, unlike recombinant affinity reagents. A recombinant scaffold based on the naturally occurring Forkhead associated (FHA) domain, which binds phosphothreonines in cellular proteins, has the potential to be a highly selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), have been isolated from phage libraries displaying an engineered form of the yeast Rad1 FHA1 domain through affinity selection with 15-mer phosphopeptides corresponding to human mTOR (mechanistic target of rapamycin), Chk2 (CHK2 checkpoint homolog), 53BP1 (tumor suppressor p53-binding protein), and AKT1 (oncogenic AGC kinase). The binding of the pTBDs is specific for phosphothreonine, as they do not bind to the same peptide sequence containing either threonine or phosphoserine. Binding is also generally specific for the phosphopeptide used in the affinity selections. The engineered FHA domains express well in Escherichia coli as maltose-binding protein (MBP) fusions, which will permit affinity measurements in the near future.