Skip to content

Research at St Andrews

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

Research output: Contribution to journalArticle

DOI

Open Access Status

  • Embargoed (until 6/12/18)

Author(s)

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

School/Research organisations

Abstract

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.
Close

Details

Original languageEnglish
Pages (from-to)771-780
JournalProtein Engineering, Design & Selection
Volume30
Issue number12
Early online date6 Dec 2017
DOIs
StatePublished - Dec 2017

    Research areas

  • Protein engineering, Fluorescence imaging, Post-translational labeling

Discover related content
Find related publications, people, projects and more using interactive charts.

View graph of relations

Related by author

  1. A new structural class of bacterial thioester domains reveals a slipknot topology

    Miller, O. K., Banfield, M. & Schwarz-Linek, U. 25 Sep 2018 In : Protein Science. Early View, 10 p.

    Research output: Contribution to journalArticle

  2. Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

    Barski, M., Brennan, B., Miller, O. K., Potter, J. A., Vijayakrishnan, S., Bhella, D., Naismith, J. H., Elliott, R. M. & Schwarz-Linek, U. 15 Sep 2017 In : eLife. 6, 20 p., e29236

    Research output: Contribution to journalArticle

  3. An internal thioester in a pathogen surface protein mediates covalent host binding

    Walden, M., Edwards, J. M., Dziewulska, A. M., Bergmann, R., Saalbach, G., Kan, S-Y., Miller, O. K., Weckener, M., Jackson, R. J., Shirran, S. L., Botting, C. H., Florence, G. J., Rohde, M., Banfield, M. J. & Schwarz-Linek, U. 2015 In : eLife. 4, 24 p., e06638

    Research output: Contribution to journalArticle

  4. An efficient method for the in vitro production of Azol(in)e-based cyclic peptides

    Houssen, W. E., Bent, A. F., McEwan, A. R., Pieiller, N., Tabudravu, J., Koehnke, J., Mann, G., Adaba, R. I., Thomas, L., Hawas, U. W., Liu, H., Schwarz-Linek, U., Smith, M. C. M., Naismith, J. H. & Jaspars, M. 15 Dec 2014 In : Angewandte Chemie International Edition. 53, 51, p. 14171-14174 4 p.

    Research output: Contribution to journalArticle

ID: 251803814