TY - JOUR
T1 - Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold
AU - Carrasco-López, César
AU - Zhao, Evan M.
AU - Gil, Agnieszka A.
AU - Alam, Nathan
AU - Toettcher, Jared E.
AU - Avalos, José L.
N1 - Funding Information:
We thank all members of Avalos and Toettcher labs for helpful comments. We also thank the Biophysics Core Facility and especially Venu Vandavasi for help with the bio-layer interferometry measurements. A.A.G. was supported by NIH Fellowship F32GM128304. J.E.T. was supported by NIH grant DP2EB024247 and J.L.A. was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research Award Number DE-SC0019363, the NSF CAREER Award CBET-1751840, The Pew Charitable Trusts, The Eric and Wendy Schmidt Transformative Technology Fund Award, and the Camille Dreyfus Teacher-Scholar Award.
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-020-17837-7
DO - 10.1038/s41467-020-17837-7
M3 - Article
C2 - 32792484
AN - SCOPUS:85089412050
SN - 2041-1723
VL - 11
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 4045
ER -