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

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

Author information

1
Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, 14853, USA.
2
Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA.
3
Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, 14853, USA.
4
Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
5
Department of Biochemistry, University of Zürich, 8057, Zürich, Switzerland.
6
Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
7
Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
8
Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.

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.

PMID:
31015433
PMCID:
PMC6478843
DOI:
10.1038/s41467-019-09854-y
[Indexed for MEDLINE]
Free PMC Article

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