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

Research output: Contribution to journalArticlepeer-review


M. Hinrichsen, M. Lenz, J. M. Edwards, O. K. Miller, S. G. J. Mochrie, P. S. Swain, U. Schwarz-Linek, L. Regan

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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.


Original languageEnglish
Pages (from-to)771-780
JournalProtein Engineering, Design & Selection
Issue number12
Early online date6 Dec 2017
Publication statusPublished - Dec 2017

    Research areas

  • Protein engineering, Fluorescence imaging, Post-translational labeling

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