A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins
| Autor: | Erin A. Stephens, Lutz Kummer, Andreas Plückthun, Fabian Brandl, Dujduan Waraho-Zhmayev, Matthew P. DeLisa, Allen Jiang, Morgan B. Ludwicki, Bunyarit Meksiriporn, Hyeon-Cheol Lee |
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| Přispěvatelé: | University of Zurich, DeLisa, Matthew P |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
High-throughput screening Recombinant Fusion Proteins Science Protein design General Physics and Astronomy 610 Medicine & health 1600 General Chemistry 02 engineering and technology Computational biology Protein Engineering DNA-binding protein General Biochemistry Genetics and Molecular Biology Article beta-Lactamases Substrate Specificity Applied microbiology 03 medical and health sciences 1300 General Biochemistry Genetics and Molecular Biology 10019 Department of Biochemistry Protein phosphorylation Phosphorylation lcsh:Science Mitogen-Activated Protein Kinase 1 Multidisciplinary Chemistry Escherichia coli Proteins General Chemistry Protein engineering 021001 nanoscience & nanotechnology 3100 General Physics and Astronomy Ankyrin Repeat 030104 developmental biology DARPin 570 Life sciences biology Ankyrin repeat lcsh:Q Target protein 0210 nano-technology Carrier Proteins Protein Processing Post-Translational |
| Zdroj: | Nature Communications, Vol 10, Iss 1, Pp 1-10 (2019) Nature Communications |
| ISSN: | 2041-1723 |
| Popis: | 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. Protein phosphorylation helps to control many important cellular activities. Here the authors describe a genetic selection strategy to isolate designed ankyrin repeat proteins that bind specifically to phosphomodified targets. |
| Databáze: | OpenAIRE |
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