Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold. Author César Carrasco-López, Evan Zhao, Agnieszka Gil, Nathan Alam, Jared Toettcher, José Avalos Publication Year 2020 Type Journal Article Abstract Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light. Keywords Escherichia coli, Humans, Protein Binding, Light, HEK293 Cells, Proteins, Optogenetics, Chromatography, Affinity Journal Nat Commun Volume 11 Issue 1 Pages 4045 Date Published 2020 Aug 13 ISSN Number 2041-1723 DOI 10.1038/s41467-020-17837-7 Alternate Journal Nat Commun PMCID PMC7427095 PMID 32792484 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML