Antibodies are incredibly powerful tools in basic research and clinical testing because they offer exquisite specificity and affinity in detecting proteins of interest in complex mixtures. They 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. Bacteriophage M13 libraries will be built that display four different FHA domains with different recognition properties for the purpose of isolating affinity reagents through affinity selection with phosphopeptides corresponding to five human proteins involved in biomedically important cell signalling pathways. The engineered FHA domains, termed phosphothreonine-biding domains (pTBDs), which have been isolated from the phage libraries through affinity selection with the phosphopeptides, will be validated in two novel manners: a) western blotting to synthetic phosphopeptides ligated to the C-terminus of maltose binding protein (MBP) and b) binding to conformationally-folded target proteins that carry phosphothreonine at defined sites. The on- rates, off-rates, and dissociation constants of the pTBDs will be measured by surface plasmon resonance (SPR) for phosphorylated and non-phosphorylated forms of the target proteins. To demonstrate the pTBDs can selectively bind their targets in complex mixtures, we will spike E. coli and commercial HeLa cell extracts with a range of concentrations of the phosphothreonine- incorporated targets and monitor the quantitative pull-down of the targets from complex mixtures by western blotting with commercial anti-target antibodies. Successful completion of the proposed experiments will lead to development of a pipeline for generating high-quality affinity reagents to phosphothreonine-based epitopes of native proteins. -1-