title: Two-strain, cell-selective protein labeling in mixed bacterial cultures.
abstract: Cell-selective metabolic labeling of proteins with noncanonical amino acids enables the study of proteomic changes in specified subpopulations of complex multicellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Truong
authors.forename: Tae Hyeon
authors.initials: TH
authors.lastname: Yoo
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lampo
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000480:Alkynes / Q000378:metabolism*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis*; D005998:Glycine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D040901:Proteomics / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000480:Alkynes; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C009055:propargylglycine; D008718:Methionine-tRNA Ligase; D005998:Glycine