An Engineered Survival-Selection Strategy for Synthetic Binding Scaffolds Specifically Targeting Post-Translationally Phosphorylated Proteins

Protein phosphorylation plays an important role in the regulation of protein function and many cellular processes. Aberrant phosphorylation has been shown to be a cause of cell death as well as maligination. As such, there is an urgent need for affinity reagents that target phospho-modified sites on individual proteins for either immunodetection or immunotherapy; however, generating such reagents remains a significant challenge. Here, we described a genetic selection strategy termed PhLI-TRAP (phospho-functional ligand-binding identification by Tat-based recognition of associating proteins) for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). PhLI-TRAP was successfully used to improve the affinity of a phospho-specific DARPin for its cognate pERK2 antigen as well as to reprogram the specificity of the same parental DARPin for binding to noncognate ERK2. Importantly, by linking antibiotic resistance with phospho-epitope binding in the cytoplasm of E. coli cells, the PhLI-TRAP method eliminates the need for purification or immobilization of the phosphoprotein target and only requires selective plating of bacteria on solid medium to uncover productive binders. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.