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Site-specific protein labeling in cells

To track protein expression, localization, activity, interactions, or conformational changes as components of cellular signaling pathways, biologists need general tools for in vivo site-specific labeling of proteins with fluorophores and other useful probes. Traditional chemical labeling methods, such as cysteine-maleimide conjugation, are too promiscuous for in vivo use, and the most widely used genetic method, fusion to green fluorescent protein (GFP), carries a payload of 238 amino acids and limits detection sensitivity due to GFP's dim fluorescence and tendency to photobleach. We are using protein engineering techniques in combination with small-molecule synthesis to develop reagents for the site-specific labeling of any desired protein in vivo. These reagents should allow the productive study of signaling events via attachment of a wide range of biophysical probes (e.g., fluorophores and photoactivatable crosslinkers) to specific protein targets in living cells.

Related publications:

Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells.
Journal of the American Chemical Society 2012, 134, 792-795.
D. S. Liu, A. Tangpeerachaikul, R. Selvaraj, M. T. Taylor, J. M. Fox, and A. Y. Ting.

A fluorophore ligase for site-specific protein labeling inside living cells.
Proceedings of the National Academy of Sciences 2010, 107, 10914-10919.
C. Uttamapinant, K. A. White, H. Baruah, S. Thompson, M. Fernández-Suárez, S. Puthenveetil, and A. Y. Ting.

Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.
Nature Biotechnology 2007, 25, 1483-1487.
M. Fernández-Suárez, H. Baruah, L. Martínez-Hernández, K. T. Xie, J. M. Baskin, C. R. Bertozzi, and A. Y. Ting.

An engineered aryl azide ligase for site-specific mapping of protein-protein interactions through photocrosslinking.
Angewandte Chemie International Edition
2008, 47, 7018-7021.
H. Baruah, S. Puthenveetil, Y.-A. Choi, S. Shah, and A. Y. Ting.

Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
Nature Methods 2005, 2, 99-104.
I. Chen, M. Howarth, W. Lin, and A. Y. Ting.

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