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Article: Rapid labeling of intracellular His-tagged proteins in living cells

TitleRapid labeling of intracellular His-tagged proteins in living cells
Authors
Issue Date2015
PublisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.org
Citation
Proceedings of the National Academy of Sciences, 2015, v. 112 n. 10, p. 2948-2953 How to Cite?
AbstractSmall molecule-based fluorescent probes have been used for realtime visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to bemainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of Histagged proteins in various prokaryotic and eukaryotic cells. © 2015, National Academy of Sciences. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/211622
ISSN
2015 Impact Factor: 9.423
2015 SCImago Journal Rankings: 6.883
PubMed Central ID
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorLAI, YT-
dc.contributor.authorCHANG, YY-
dc.contributor.authorHu, L-
dc.contributor.authorYANG, Y-
dc.contributor.authorCHAO, A-
dc.contributor.authorDu, Z-
dc.contributor.authorTanner, JA-
dc.contributor.authorChye, ML-
dc.contributor.authorQian, C-
dc.contributor.authorNg, KM-
dc.contributor.authorSun, H-
dc.date.accessioned2015-07-21T02:05:23Z-
dc.date.available2015-07-21T02:05:23Z-
dc.date.issued2015-
dc.identifier.citationProceedings of the National Academy of Sciences, 2015, v. 112 n. 10, p. 2948-2953-
dc.identifier.issn0027-8424-
dc.identifier.urihttp://hdl.handle.net/10722/211622-
dc.description.abstractSmall molecule-based fluorescent probes have been used for realtime visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to bemainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of Histagged proteins in various prokaryotic and eukaryotic cells. © 2015, National Academy of Sciences. All rights reserved.-
dc.languageeng-
dc.publisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.org-
dc.relation.ispartofProceedings of the National Academy of Sciences-
dc.rightsProceedings of the National Academy of Sciences. Copyright © National Academy of Sciences.-
dc.titleRapid labeling of intracellular His-tagged proteins in living cells-
dc.typeArticle-
dc.identifier.emailTanner, JA: jatanner@hkucc.hku.hk-
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hk-
dc.identifier.emailQian, C: cmqian@hku.hk-
dc.identifier.emailNg, KM: kwanmng@hku.hk-
dc.identifier.emailSun, H: hsun@hku.hk-
dc.identifier.authorityTanner, JA=rp00495-
dc.identifier.authorityChye, ML=rp00687-
dc.identifier.authorityQian, C=rp01371-
dc.identifier.authorityNg, KM=rp00766-
dc.identifier.authoritySun, H=rp00777-
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1073/pnas.1419598112-
dc.identifier.pmid25713372-
dc.identifier.pmcidPMC4364221-
dc.identifier.scopuseid_2-s2.0-84924309930-
dc.identifier.hkuros245256-
dc.identifier.volume112-
dc.identifier.issue10-
dc.identifier.spage2948-
dc.identifier.epage2953-
dc.identifier.isiWOS:000350646500029-
dc.publisher.placeUnited States-
dc.identifier.f1000725367569-

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