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

Bunyarit Meksiriporn, Morgan B. Ludwicki, Erin A. Stephens, Allen Jiang, Hyeon-Cheol Lee, Dujduan Waraho-Zhmayev, Lutz Kummer, Fabian Brandl, Andreas Plückthun and Matthew P. DeLisa ()
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Bunyarit Meksiriporn: Cornell University
Morgan B. Ludwicki: Cornell University
Erin A. Stephens: Cornell University
Allen Jiang: Cornell University
Hyeon-Cheol Lee: Cornell University
Dujduan Waraho-Zhmayev: King Mongkut’s University of Technology Thonburi
Lutz Kummer: University of Zürich
Fabian Brandl: University of Zürich
Andreas Plückthun: University of Zürich
Matthew P. DeLisa: Cornell University

Nature Communications, 2019, vol. 10, issue 1, 1-10

Abstract: Abstract There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy 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). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. 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.

Date: 2019
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DOI: 10.1038/s41467-019-09854-y

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