Fig. 1 | Nature Communications

Fig. 1

From: A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins

Fig. 1

PhLI-TRAP-based isolation of phospho-specific binding proteins. Schematic representation of engineered assay for co-translocation of interacting receptor-antigen pairs via the Tat translocase (TatABC). The assay enables discovery and optimization of synthetic binding proteins (e.g., DARPins) with affinity for phospho-modified target antigens simply by demanding bacterial growth on β-lactam antibiotics such as carbenicillin (Carb), without the need for purification or immobilization of the phosphoprotein target. The Tat signal peptide chosen was spTorA, the reporter enzyme was Bla, the synthetic binding protein was an ERK2- or pERK2-specific DARPin, and the antigen was ERK2. Phosphorylation status of ERK2 was toggled by expression of the constitutively active upstream kinase MEK1R4F, which doubly phosphorylates (yellow P circles) ERK2 in the cytoplasm of living E. coli cells

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