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

Nat Commun. 2019 Apr 23;10(1):1830. doi: 10.1038/s41467-019-09854-y.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ankyrin Repeat / genetics
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Escherichia coli Proteins / genetics
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Phosphorylation
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational*
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity / genetics
  • beta-Lactamases / genetics


  • Carrier Proteins
  • Escherichia coli Proteins
  • Recombinant Fusion Proteins
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • beta-Lactamases
  • beta-lactamase TEM-1