Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry
- 10 June 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 131 (25), 8720-8721
- https://doi.org/10.1021/ja900553w
Abstract
We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNACUA pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-l-lysine, containing a carbon−carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-l-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddtion reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.Keywords
This publication has 16 references indexed in Scilit:
- Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein ModificationCell Chemical Biology, 2008
- Genetically encoding Nε-acetyllysine in recombinant proteinsNature Chemical Biology, 2008
- Pyrrolysine is not hardwired for cotranslational insertion at UAG codonsProceedings of the National Academy of Sciences of the United States of America, 2007
- A chemical toolkit for proteins — an expanded genetic codeNature Reviews Molecular Cell Biology, 2006
- In vivo incorporation of an alkyne into proteins in Escherichia coliBioorganic & Medicinal Chemistry Letters, 2005
- An Expanded Eukaryotic Genetic CodeScience, 2003
- A New Strategy for the Site-Specific Modification of Proteins in VivoBiochemistry, 2003
- Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coliJournal of the American Chemical Society, 2002
- A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in Vivo Incorporation of Aryl Ketone Functionality into ProteinsJournal of the American Chemical Society, 2002
- Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligationProceedings of the National Academy of Sciences of the United States of America, 2001