In-cell covalent labeling of reactive His-tag fused proteins

Uchinomiya, Shohei; Nonaka, Hiroshi; Wakayama, Sho; Ojida, Akio; Hamachi, Itaru

doi:10.1039/c3cc41979g

Cited by 49 publications

(57 citation statements)

References 29 publications

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“…The perturbation of protein localization may be attributed to the relatively large size of RFP (27.5 kDa) (5) compared with XPA122 (15 kDa). By contrast, using the Ni-NTA-AC probe, His-XPA122 was found to be enriched in the nucleus, in agreement with the previously reported intracellular localization of XPA, a protein involved in the recognition of DNA damage during nucleotide excision repair processes (16,29). Taken together, we demonstrate that the new fluorescent probe Ni-NTA-AC can be preferentially applied to track the abundances and localization of His-tagged proteins in live mammalian cells, in particular for small proteins.…”

Section: Evaluation Of Ni-nta-ac Probe For Labeling Of His-tagged Prosupporting

confidence: 73%

“…Recently a di-NTA-based fluorescent probe containing an α-chloroacetamide that targets the Cys-appended His-tag (CysHis 6 -tag) was reported to be able to label intracellular Cys-His 6 -tagged proteins in live cells (16). However, the di-NTA fluorophore conjugate itself could hardly enter cells, unless the conjugate was attached to a cargo consisting of a cell-penetrating peptide and fluorescent quencher (a dabsyl-appended icosapeptide with a dabsyl group, a tetra-His, and an octa-Arg), enforcing the probe to enter cells to label Cys-His 6 -tagged instead of His 6 -tagged proteins.…”

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confidence: 99%

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Rapid labeling of intracellular His-tagged proteins in living cells

Lai¹,

Chang²,

Hu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Small molecule-based fluorescent probes have been used for real-time 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 be mainly 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 His-tagged proteins in various prokaryotic and eukaryotic cells.

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Section: Evaluation Of Ni-nta-ac Probe For Labeling Of His-tagged Prosupporting

confidence: 73%

mentioning

confidence: 99%

Rapid labeling of intracellular His-tagged proteins in living cells

Lai¹,

Chang²,

Hu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…CysN i-gerichtetes Elektrophil [156] Ni eGFP (27) 6.2 LysN i-gerichtetes Elektrophil [216] Ni Transferrin (77) 6.2 viele…”

Section: Proximitätsvermitteltunclassified

“…Obwohl sie auf eine einzige Gruppe an Funktionalisierungen beschränkt ist, hat sich die Azidgruppe zu einem essenziellen Bestandteil der gängigen Biokonjugationsmethoden entwickelt und die oben beschriebene Te chnik wurde bereits von verschiedenen Arbeitsgruppen ausführlich angewendet. Gleichermaßen fallen auch Technologien zur Affinitätsidentifikation durch Metalle,w ie etwa Nickel/His-Tags [154][155][156] oder Zink/Tetraaspartat-Paare, [157][158][159] weitestgehend aus seinem Fokus heraus,obwohl sie fürAnwendungen zur Aufreinigung oder Detektion von Nutzen sein kçnnen. [138] Kürzlich wurde außerdem eine reaktionszentrenselektive Version der Azidtransferreaktion an Avidinderivaten durch die Inkorporation eines Biotinzentrums in den Imidazolring erreicht.…”

Section: Angewandte Chemieunclassified

Metallvermittelte Funktionalisierung natürlicher Peptide und Proteine: Biokonjugation mit Übergangsmetallen

2019

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Die selektive Modifikation natürlicher Proteine stellt eine enorme methodologische Herausforderung dar und bedarf einer strikten Kontrolle der Selektivität und des Umfangs einer Reaktion. Demnach existiert ein fortwährender Bedarf an neuen Reaktivitäts‐ sowie Selektivitätskonzepten. Übergangsmetalle zeichnen sich durch ihre Fülle einzigartiger Reaktivitäten aus, welche biologischen Reaktionen und Prozessen gegenüber orthogonal sind. Folglich spielen metallbasierte Methoden eine zunehmend wichtige Rolle in der Biokonjugationschemie. In diesem Aufsatz werden metallbasierte Methoden sowie deren Reaktivitäten und Selektivitäten bei der Funktionalisierung natürlicher Proteine und Peptide diskutiert.

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“…About 50% labeling efficiency was observed after 20 min (Figure 2c), and after 100 min, the labeling yield was determined to be 84%. The second-order rate constant was calculated as 1100 M −1 s −1 ( Figure S2), which is comparable to previously reported protein labeling reactions based on a reactive His-tag, 21 CLIP-tag, 4 or on a tetrazine ligation, 32 a fast bio-orthogonal click reaction. However, it is about ∼25-and ∼65-fold slower than SNAP-tag 4 and FlAsH-tag 17 based labeling reactions, respectively.…”

mentioning

confidence: 99%

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag

2015

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Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

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In-cell covalent labeling of reactive His-tag fused proteins

Cited by 49 publications

References 29 publications

Rapid labeling of intracellular His-tagged proteins in living cells

Rapid labeling of intracellular His-tagged proteins in living cells

Metallvermittelte Funktionalisierung natürlicher Peptide und Proteine: Biokonjugation mit Übergangsmetallen

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag

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