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dc.contributor.authorHinrichsen, M.
dc.contributor.authorLenz, M.
dc.contributor.authorEdwards, J. M.
dc.contributor.authorMiller, O. K.
dc.contributor.authorMochrie, S. G. J.
dc.contributor.authorSwain, P. S.
dc.contributor.authorSchwarz-Linek, U.
dc.contributor.authorRegan, L.
dc.date.accessioned2018-12-06T00:48:30Z
dc.date.available2018-12-06T00:48:30Z
dc.date.issued2017-12
dc.identifier.citationHinrichsen , 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/gzx059en
dc.identifier.issn1741-0126
dc.identifier.otherPURE: 251803814
dc.identifier.otherPURE UUID: cd76b075-6d06-4330-a9de-1f40bb0a3b00
dc.identifier.otherPubMed: 29228311
dc.identifier.otherScopus: 85041616032
dc.identifier.otherORCID: /0000-0003-0526-223X/work/40714979
dc.identifier.otherWOS: 000425541500001
dc.identifier.urihttp://hdl.handle.net/10023/16634
dc.descriptionThis 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.abstractWe 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.language.isoeng
dc.relation.ispartofProtein Engineering, Design & Selectionen
dc.rights© The Author 2017. Published by Oxford University Press. This work has been made available online in accordance with the publisher’s policies. This is the author created, accepted version manuscript following peer review and may differ slightly from the final published version. The final published version of this work is available at https://doi.org/10.1093/protein/gzx059en
dc.subjectProtein engineeringen
dc.subjectFluorescence imagingen
dc.subjectPost-translational labelingen
dc.subjectQD Chemistryen
dc.subjectQH301 Biologyen
dc.subjectBiochemistryen
dc.subjectNDASen
dc.subject.lccQDen
dc.subject.lccQH301en
dc.titleA new method for post-translationally labeling proteins in live cells for fluorescence imaging and trackingen
dc.typeJournal articleen
dc.description.versionPostprinten
dc.contributor.institutionUniversity of St Andrews.Biomedical Sciences Research Complexen
dc.contributor.institutionUniversity of St Andrews.School of Biologyen
dc.identifier.doihttps://doi.org/10.1093/protein/gzx059
dc.description.statusPeer revieweden
dc.date.embargoedUntil2018-12-06
dc.identifier.urlhttps://academic.oup.com/peds/article/30/12/771/4710354#supplementary-dataen


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