Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells
Abstract
Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.
Additional Information
© 2013 National Academy of Sciences. Edited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved February 11, 2013 (received for review September 19, 2012). Published online before print March 11, 2013. We thank Michael Sweredowski, Robert Graham, and Sonja Hess of the Proteome Exploration Laboratory (Beckman Institute, Caltech) for their help and advice in collecting and analyzing tandem mass spectrometry data; Angela Ho and Jost Vielmetter of the Protein Expression Center (Beckman Institute, Caltech) for their help in generating stable cell lines; Carolyn Bertozzi and her laboratory (University of California, Berkeley) for their gift of DIFOAlexa Fluor 488; and John Phillips (Caltech) and members of D.A.T.'s laboratory for helpful discussions. Support for this work was provided by National Institutes of Health Grant R01 GM062523, the Joseph J. Jacobs Institute for Molecular Engineering for Medicine, and the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the US Army Research Office. Author contributions: J.T.N. and D.A.T. designed research; J.T.N. performed research; J.T.N., E.M.S., and D.A.T. analyzed data; and J.T.N., E.M.S., and D.A.T. wrote the paper.Attached Files
Published - PNAS-2013-Ngo-4992-7.pdf
Supplemental Material - Appendix.pdf
Supplemental Material - Dataset.pdf
Files
Additional details
- PMCID
- PMC3612679
- Eprint ID
- 38775
- Resolver ID
- CaltechAUTHORS:20130604-100505112
- R01 GM062523
- NIH
- Joseph J. Jacobs Institute for Molecular Engineering for Medicine
- W911NF-09-0001
- Army Research Office (ARO)
- Created
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2013-06-05Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field