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A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking
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dc.contributor.author | Hinrichsen, M. | |
dc.contributor.author | Lenz, M. | |
dc.contributor.author | Edwards, J. M. | |
dc.contributor.author | Miller, O. K. | |
dc.contributor.author | Mochrie, S. G. J. | |
dc.contributor.author | Swain, P. S. | |
dc.contributor.author | Schwarz-Linek, U. | |
dc.contributor.author | Regan, L. | |
dc.date.accessioned | 2018-12-06T00:48:30Z | |
dc.date.available | 2018-12-06T00:48:30Z | |
dc.date.issued | 2017-12 | |
dc.identifier | 251803814 | |
dc.identifier | cd76b075-6d06-4330-a9de-1f40bb0a3b00 | |
dc.identifier | 29228311 | |
dc.identifier | 85041616032 | |
dc.identifier | 000425541500001 | |
dc.identifier.citation | Hinrichsen , M , Lenz , M , Edwards , J M , Miller , O K , Mochrie , S G J , Swain , P S , Schwarz-Linek , U & Regan , L 2017 , ' A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking ' , Protein Engineering, Design & Selection , vol. 30 , no. 12 , pp. 771-780 . https://doi.org/10.1093/protein/gzx059 | en |
dc.identifier.issn | 1741-0126 | |
dc.identifier.other | ORCID: /0000-0003-0526-223X/work/40714979 | |
dc.identifier.uri | https://hdl.handle.net/10023/16634 | |
dc.description | This work was funded by the Raymond and Beverley Sackler Institute for Biological, Physical, and Engineering sciences [to L.R.]; the National Institute of Health [Grant nos. GM118528 and CA209992 to M. H. and L. R.]; the Medical Research Council [Grant no. MR/K001485 to U.S.L. and J. M. E.]; a Leverhulme Trust Visiting Professorship [to L. R.]; and Royal Society of Edinburgh [Caledonian Scholarship to O.K.M.]. | en |
dc.description.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. | |
dc.format.extent | 8227974 | |
dc.language.iso | eng | |
dc.relation.ispartof | Protein Engineering, Design & Selection | en |
dc.subject | Protein engineering | en |
dc.subject | Fluorescence imaging | en |
dc.subject | Post-translational labeling | en |
dc.subject | QD Chemistry | en |
dc.subject | QH301 Biology | en |
dc.subject | Biochemistry | en |
dc.subject | NDAS | en |
dc.subject.lcc | QD | en |
dc.subject.lcc | QH301 | en |
dc.title | A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking | en |
dc.type | Journal article | en |
dc.contributor.sponsor | Medical Research Council | en |
dc.contributor.institution | University of St Andrews. Biomedical Sciences Research Complex | en |
dc.contributor.institution | University of St Andrews. School of Biology | en |
dc.identifier.doi | https://doi.org/10.1093/protein/gzx059 | |
dc.description.status | Peer reviewed | en |
dc.date.embargoedUntil | 2018-12-06 | |
dc.identifier.url | https://academic.oup.com/peds/article/30/12/771/4710354#supplementary-data | en |
dc.identifier.grantnumber | MR/K001485/1 | en |
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