title: A genetically encoded and gate for cell-targeted metabolic labeling of proteins.
abstract: We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors: Alborz
authors: A
authors: Mahdavi
authors: Thomas H
authors: TH
authors: Segall-Shapiro
authors: Songzi
authors: S
authors: Kou
authors: Granton A
authors: GA
authors: Jindal
authors: Kevin G
authors: KG
authors: Hoff
authors: Shirley
authors: S
authors: Liu
authors: Mohsen
authors: M
authors: Chitsaz
authors: Rustem F
authors: RF
authors: Ismagilov
authors: Jonathan J
authors: JJ
authors: Silberg
authors: David A
authors: DA
authors: Tirrell
mesh_terms: D002384:Catalysis; D003500:Cyclization; D005818:Genetic Engineering; D008718:Methionine-tRNA Ligase / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D008718:Methionine-tRNA Ligase