Figure 4.

(Top) Cell-selective BONCAT performed in a mixture of cells. Restricting expression of a mutant synthetase to a certain cell restricts Anl labeling to that cell (highlighted in blue). Proteins synthesized in cells (highlighted in gray) that do not express the mutant synthetase are neither labeled nor detected following enrichment. (Bottom) Cell-selective labeling in mixtures of bacterial and mammalian cells. (a) In Anl-containing mixed cultures of E. coli and mouse alveolar macrophages, only E. coli cells constitutively expressing the mutant NLL-EcMetRS were labeled by TAMRA-alkyne. Macrophages were labeled with Mitotracker Deep Red and displayed low TAMRA-alkyne background emission. (b) In Aha-containing mixed cultures of E. coli and mouse alveolar macrophages, both wild-type E. coli cells and macrophages exhibited strong TAMRA-alkyne emission; incorporation of Aha occurs in both cell types. (c) Mixed cell lysate was subjected to conjugation with alkyne-functionalized biotin, and labeled proteins were enriched by NeutrAvidin affinity chromatography. Immunoblotting of unbound flow-through (FT), washes (W1, W3, W5) and eluent (E) reveals enrichment of the bacterial marker protein GFP. (Adapted with permission from Ngo et al., Nat. Chem. Biol., 5, 715–717, 2009. Copyright 2009 Nature Publishing Group.)