Ligand-directed tosyl chemistry for protein labeling in vivo

Tsukiji, Shinya; Miyagawa, Masayoshi; Takaoka, Yousuke; Tamura, Tomonori; Hamachi, Itaru

doi:10.1038/nchembio.157

Cited by 337 publications

(293 citation statements)

References 25 publications

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“…Whereas all previously discussed protein tags rely on genetic manipulations, namely the expression of the POI as fusion to the particular protein tag, Tsukiji et al reported a native protein labeling strategy [29]. In this method, the ligand binds with high affinity to the native target protein directing a reactive group to the surface of the protein that forms a covalent bond through proximity-induced reactivity.…”

Section: Proximity Induced Covalent Protein Tagsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Proximity Induced Covalent Protein Tagsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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“…By using a ligand-directed, S N 2 reactive, tosyl group bound to the probe of interest, the ligand is released on conjugation and so does not compromise protein function 193 . The amino acids modified by this technique include non-traditional nucleophilic amino acids such as histidine and glutamic acid, strongly suggesting this as a reaction driven by proximity, rather than nucleophilicity 194 .…”

Section: Box 2 | Comparing Strategies For Positional Controlmentioning

confidence: 99%

Selective chemical protein modification

2014

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“…The molecule mediating the interaction is released when the covalent coupling of the label to the protein takes place, which reduces its impact on protein function compared to previous approaches. This method has been shown to allow selective native protein labeling in the cell interior and even in blood cells of living animals [73 ]. The method is remarkably specific, but the general reactivity of the tosyl group with nucleophiles may inherently limit the attainable selectivity towards proteins of low abundance.…”

Section: Conclusion and Future Developmentsmentioning

confidence: 99%

“…A less invasive route has been described by Hamachi and co-workers and is based -similar to their self-labeling tag approach (see above) -on turning an initially noncovalent interaction into a covalent one. Here, the noncovalent interaction is provided by a molecule that specifically binds to the native protein of interest, for example, an enzyme inhibitor [73 ]. The reactive group used is the tosyl group, which is why this approach has been termed liganddirected tosyl chemistry (LDT).…”

Section: Conclusion and Future Developmentsmentioning

confidence: 99%

How to obtain labeled proteins and what to do with them

Hinner

Johnsson

2010

Current Opinion in Biotechnology

258

217

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We review new and established methods for the chemical modification of proteins in living cells and highlight recent applications. The review focuses on tag-mediated protein labeling methods, such as the tetracysteine tag and SNAP-tag, and new developments in this field such as intracellular labeling with lipoic acid ligase. Recent promising advances in the incorporation of unnatural amino acids into proteins are also briefly discussed. We describe new tools using tag-mediated labeling methods including the super-resolution microscopy of tagged proteins, the study of the interactions of proteins and protein domains, the subcellular targeting of synthetic ion sensors, and the generation of new semisynthetic metabolite sensors. We conclude with a view on necessary future developments, with one example being the selective labeling of non-tagged, native proteins in complex protein mixtures. IntroductionChemists are becoming increasingly fascinated with derivatizing proteins by genetically non-encodable synthetic molecules. As discussed in this review, such molecules include fluorescent dyes, chemical crosslinkers, pharmacologically active compounds, and synthetic fluorescent probes for ions such as Ca 2+ . The labeling of proteins with synthetic molecules provides exciting new tools for studying proteins and their function in the cell, and is promising to have a strong impact on drug development. In this review, we will provide a concise overview over established and new methods that provide proteins derivatized with a label, and give illustrative recent examples of their application. For further information, the reader is directed to recently published more exhaustive reviews [1][2][3][4]. The focus of our review lies on covalent tag-based labeling methods, as these allow an efficient and irreversible transfer of labels in mammalian cells. However, we will also comment on unnatural amino acid incorporation as a tool of increasing importance for mammalian cell studies. The discussion of methods and applications is preceded by general considerations on tag-mediated labeling. We will conclude with an outlook on future directions and highlight areas that would profit most from new developments. Tag-mediated labelingAn ideal method for tag-based protein labeling should exhibit the following features: (i) the possibility to introduce any label of choice in one step, (ii) fast and quantitative labeling, (iii) no labeling of non-target proteins, (iv) a small tag to minimize its impact on protein function, (v) the formation of a stable, covalent bond between protein and label, and (vi) no side effects of the reagents used for labeling. Finally, an ideal tag should work in vitro, in complex protein samples, on the cell surface, within the cell and cellular compartments, and in living animals (in vivo), with this order representing an increasing level of difficulty. None of the existing labeling methods fulfils all these requirements. It is notable, however, that the existing methods of tag-mediated labeling can be grouped ...

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Ligand-directed tosyl chemistry for protein labeling in vivo

Cited by 337 publications

References 25 publications

Chemical tags: Applications in live cell fluorescence imaging

Chemical tags: Applications in live cell fluorescence imaging

Selective chemical protein modification

How to obtain labeled proteins and what to do with them

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