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Protein Eng Des Sel. 2017 Dec 1;30(12):771-780. doi: 10.1093/protein/gzx059.

A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking.

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Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT06511, USA.
SynthSys-Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK.
Biomedical Sciences Research Complex and School of Biology, University of St Andrews, North Haugh, St AndrewsKY16 9ST, UK.
Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06511, USA.
Department of Physics, Yale University, 217 Prospect St, New Haven, CT 06511, USA.
Department of Applied Physics, Yale University, 15 Prospect Street, New Haven, CT 06511, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA.
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, 06511, USA.


We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.


S. cerevisiae; SpyCatcher-SpyTag; membrane protein; protein engineering; single cell

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