Reporting in Nature Chemical Biology, Ngo et al. describe a system for cell-selective protein labelling in mixtures of cells (J. T. Ngo et al. Nature Chem. Biol. doi:10.1038/nchembio.200; 2009). Their work is based on the principle that proteins can be tracked in experiments if they are engineered so that some of their constituent amino acids contain a tag, such as a radioactive label; tagged amino acids can be added to cells in culture, whereupon the cells incorporate them into newly formed proteins. The general problem with this approach is that all the cells in the culture become labelled, whereas it might be that only certain types of cell need to be tagged.
Ngo and colleagues' solution to that problem involves non-naturally occurring amino acids that contain azide (N3) groups in their side chains. Azides don't react with biological molecules, but under certain conditions they do react quickly with synthetic molecules that contain alkyne groups (which have carbon–carbon triple bonds). Once incorporated into proteins, azide-containing amino-acid residues will thus react with alkyne-containing fluorescent dyes or affinity reagents, so tagging the proteins for imaging, detection or separation.
Non-natural amino acids are already used for protein labelling, but Ngo et al. take the idea further. They exploit an azide-containing amino acid that is incorporated into proteins only by cells that express a mutant of the methionyl-tRNA synthetase enzyme (which is involved in protein synthesis). When the authors added this amino acid to a co-culture of normal and mutant Escherichia coli cells, only the mutants were subsequently tagged with an alkyne-containing affinity reagent or fluorescent dye.
To show that their technique could be applied to mixtures of bacterial and mammalian cells, Ngo et al. infected mouse macrophages with E. coli cells that express the mutant enzyme, in the presence of the azide-containing amino acid. They then treated the macrophages with a green alkyne-containing dye, which tagged only the bacterial cells (pictured; macrophages are stained orange, and are about 15 micrometres in diameter). The authors were also able to specifically tag newly synthesized bacterial proteins with an affinity reagent, which was then used as a handle to isolate those proteins from the culture.
The authors' approach allows the cellular origins of proteins in complex multicellular systems to be determined. It could therefore be handy for isolating proteins from pathogens in studies of infections, for example, or for identifying the complement of proteins of a single bacterial species living in a community of many other microbial organisms.