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Published September 17, 2003 | Supplemental Material
Journal Article Open

Cell Surface Labeling of Escherichia coli via Copper(I)-Catalyzed [3+2] Cycloaddition

Abstract

Labeling of the cell surface of Escherichia coli was accomplished by expression of a recombinant outer membrane protein, OmpC, in the presence of the unnatural amino acid azidohomoalanine, which acts as a methionine surrogate. The surface-exposed azide moieties of whole cells were biotinylated via Cu(1)-catalyzed [3+2] azide-alkyne cycloaddition. The specificity of labeling of both wild-type OmpC and a mutant containing additional methionine sites for azidohomoalanine incorporation was confirmed by Western blotting. Flow cytometry was performed to examine the specificity of the labeling. Cells that express the mutant form of OmpC in the presence of azidohomoalanine, which were biotinylated and stained with fluorescent avidin, exhibit a mean fluorescence 10-fold higher than the background. Incorporation of an unnatural amino acid can thus be determined on a single-cell basis.

Additional Information

Copyright © 2003 American Chemical Society. Published In Issue September 17, 2003. Publication Date (Web): August 23, 2003 Received June 18, 2003. Acknowledgment. We thank K. Barry Sharpless and Valery Fokin for their gift of the triazole ligand and for helpful advice on the azide-alkyne cycloaddition. We also thank Isaac Carrico, Pin Wang, and Rochelle Diamond for helpful discussions and Rich Roberts for use of his flow cytometer. This work was supported by the NSF Center for the Science and Engineering of Materials at Caltech. A.J.L. is an NSF Graduate Research Fellow. Supporting Information Available: Experimental protocols and details about flow cytometry (PDF).

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Created:
August 19, 2023
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October 19, 2023