A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking
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| Názov: | A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking |
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| Autori: | Hinrichsen, M., Lenz, M., Edwards, J. M., Miller, O. K., Mochrie, S. G. J., Swain, P. S., Schwarz-Linek, U., Regan, L. |
| Prispievatelia: | Medical Research Council, University of St Andrews.Biomedical Sciences Research Complex, University of St Andrews.School of Biology |
| Zdroj: | 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 : PEDS, vol. 30, no. 12, pp. 771-780 . https://doi.org/10.1093/protein/gzx059 |
| Informácie o vydavateľovi: | Oxford University Press (OUP), 2017. |
| Rok vydania: | 2017 |
| Predmety: | 0301 basic medicine, Post-translational labeling, Fluorescent Dyes/analysis, QH301 Biology, NDAS, Cell Tracking/methods, Saccharomyces cerevisiae, Protein Engineering, Biochemistry, Fluorescence imaging, Saccharomyces cerevisiae/genetics, QH301, 03 medical and health sciences, QD, Fluorescent Dyes, 0303 health sciences, Single-Cell Analysis/methods, Spectrometry, Fluorescence/methods, QD Chemistry, 3. Good health, Spectrometry, Fluorescence, Cell Tracking, Protein engineering, Single-Cell Analysis, Protein Processing, Post-Translational, Protein Engineering/methods |
| Popis: | 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. |
| Druh dokumentu: | Article |
| Popis súboru: | application/pdf |
| Jazyk: | English |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzx059 |
| Prístupová URL adresa: | https://academic.oup.com/peds/article-pdf/30/12/771/29098404/gzx059.pdf https://pubmed.ncbi.nlm.nih.gov/29228311 https://academic.oup.com/peds/article/30/12/771/4710354 https://www.ncbi.nlm.nih.gov/pubmed/29228311 http://europepmc.org/abstract/MED/29228311 https://research-repository.st-andrews.ac.uk/handle/10023/16634 https://pubmed.ncbi.nlm.nih.gov/29228311/ https://research-repository.st-andrews.ac.uk/bitstream/10023/16634/1/Hinrichsen_final.pdf https://www.pure.ed.ac.uk/ws/files/85692765/A_New_Method_for_Post_translationally_AAM_Hinrichsen_final.pdf https://hdl.handle.net/20.500.11820/3c278faa-11fa-43d6-a901-eccbfa687e99 https://hdl.handle.net/10023/16634 https://academic.oup.com/peds/article/30/12/771/4710354#supplementary-data |
| Prístupové číslo: | edsair.doi.dedup.....3af92fc881d0523eba3201b04f1f89da |
| Databáza: | OpenAIRE |
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Nájsť tento článok vo Web of Science | Abstrakt: | 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. |
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| ISSN: | 17410134 17410126 |
| DOI: | 10.1093/protein/gzx059 |