Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition.
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.
Link
A James
AJ
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.
Tirrell
David A
DA
D000409:Alanine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry* / Q000494:pharmacology*; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology* / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry; D008956:Models, Chemical; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018272:Porins / Q000737:chemistry; D013194:Staining and Labeling
D008565:Membrane Proteins; C113724:OmpC protein; D018272:Porins; C506098:azidohomoalanine; D003300:Copper; D000409:Alanine