Mahdavi, Alborz and Segall-Shapiro, Thomas H. and Kou, Songzi and Jindal, Granton A. and Hoff, Kevin G. and Liu, Shirley and Chitsaz, Mohsen and Ismagilov, Rustem F. and Silberg, Jonathan J. and Tirrell, David A. (2013) A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins. Journal of the American Chemical Society, 135 (8). pp. 2979-2982. ISSN 0002-7863. PMCID PMC3620012. doi:10.1021/ja400448f. https://resolver.caltech.edu/CaltechAUTHORS:20130404-103625840
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Abstract
We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide–alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.
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Additional Information: | © 2013 American Chemical Society. Received: January 15, 2013; Published: February 13, 2013. We are grateful for financial support by National Institutes of Health grant NIH RO1 GM062523 and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation (D.A.T.), the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office (D.A.T. and R.F.I.), the American Heart Association (J.J.S.), and the Robert A. Welch Foundation (J.J.S.). A.M. was supported by a scholarship from the National Science and Engineering Research Council of Canada and by a postgraduate scholarship from the Donna and Benjamin M. Rosen Center for Bioengineering at Caltech. | ||||||||||||||||
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Issue or Number: | 8 | ||||||||||||||||
PubMed Central ID: | PMC3620012 | ||||||||||||||||
DOI: | 10.1021/ja400448f | ||||||||||||||||
Record Number: | CaltechAUTHORS:20130404-103625840 | ||||||||||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20130404-103625840 | ||||||||||||||||
Official Citation: | A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins Alborz Mahdavi, Thomas H. Segall-Shapiro, Songzi Kou, Granton A. Jindal, Kevin G. Hoff, Shirley Liu, Mohsen Chitsaz, Rustem F. Ismagilov, Jonathan J. Silberg, and David A. Tirrell Journal of the American Chemical Society 2013 135 (8), 2979-2982 | ||||||||||||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||
ID Code: | 37763 | ||||||||||||||||
Collection: | CaltechAUTHORS | ||||||||||||||||
Deposited By: | INVALID USER | ||||||||||||||||
Deposited On: | 04 Apr 2013 18:11 | ||||||||||||||||
Last Modified: | 09 Nov 2021 23:31 |
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