Abstract
The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine–fluorophore conjugates in a bioorthogonal Diels–Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.
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Lang, K., Davis, L., Chin, J.W. (2015). Genetic Encoding of Unnatural Amino Acids for Labeling Proteins. In: Gautier, A., Hinner, M. (eds) Site-Specific Protein Labeling. Methods in Molecular Biology, vol 1266. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2272-7_15
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DOI: https://doi.org/10.1007/978-1-4939-2272-7_15
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