A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

作者：Mahdavi Alborz; Segall Shapiro Thomas H; Kou Songzi; Jindal Granton A; Hoff Kevin G; Liu Shirley; Chitsaz Mohsen; Ismagilov Rustem F; Silberg Jonathan J; Tirrell David A^*

来源：Journal of the American Chemical Society, 2013, 135(8): 2979-2982.

DOI：10.1021/ja400448f

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.

• 出版日期2013-2-27