Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line |
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| Affiliation: | 1. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois;2. Center for Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois;3. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois;4. School of Molecular and Cellular Biology, University of Leeds, Leeds, UK;5. Department of Cancer Research, Loyola University Chicago, Maywood, Illinois;6. Institute of Medical Sciences, School of Medicine Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK;7. Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, Illinois;8. Cancer Center at Illinois, University of Illinois Urbana-Champaign, Urbana, Illinois;9. Division of Nutritional Sciences, University of Illinois Urbana-Champaign, Urbana, Illinois;10. Carl R. Woese Institute for Genomic Biology, Anticancer Discovery from Pets to People Theme, University of Illinois Urbana-Champaign, Urbana, Illinois;11. University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, Illinois;12. Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago (UIC), Chicago, Illinois;13. UI Cancer Center, University of Illinois at Chicago (UIC), Chicago, Illinois;14. Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois |
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| Abstract: | Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent—an Affimer protein—that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein. |
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