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Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Ngo, John T. and Schuman, Erin M. and Tirrell, David A. (2013) Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 110 (13). pp. 4992-4997. ISSN 0027-8424. PMCID PMC3612679. https://resolver.caltech.edu/CaltechAUTHORS:20130604-100505112

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.1216375110 DOIArticle
http://www.pnas.org/content/110/13/4992PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612679/PubMed CentralArticle
ORCID:
AuthorORCID
Schuman, Erin M.0000-0002-7053-1005
Tirrell, David A.0000-0003-3175-4596
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.
Funders:
Funding AgencyGrant Number
NIHR01 GM062523
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Army Research Office (ARO)W911NF-09-0001
Subject Keywords:protein synthesis; proteomics; translational profiling; protein engineering
Issue or Number:13
PubMed Central ID:PMC3612679
Record Number:CaltechAUTHORS:20130604-100505112
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130604-100505112
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38775
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:05 Jun 2013 16:51
Last Modified:09 Mar 2020 13:19

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