(182c) Directed Evolution of An E. Coli Methionyl-tRNA Synthetase for Two Strain Cell-Selective Proteomic Labeling

Cell-selective metabolic labeling of proteins using non-canonical amino acids enables the study of proteomic changes in a single subpopulation of a complex multi-cellular environment.  Currently this approach is limited to a single amino acid analog, azidonorleucine (Anl), using an engineered methionyl-tRNA synthetase (NLL-MetRS) for incorporation at codons designated for methionine (Met).  To expand the set of tools, propargylglycine (Pra) was chosen as the target amino acid since it’s terminal alkyne side chain can undergo a copper(I)-catalyzed  [3+2] cycloaddition with azides, allowing for conjugation of fluorescence probes and affinity tags to proteins.  Using both active-site randomization and error-prone PCR, two rounds of directed evolution yielded a MetRS variant (PraRS) capable of incorporating Pra at near-quantitative level (~90%) in Met-depleted media using a Met-auxotrophic E. coli.  Proteins synthesized in E. coli strains expressing PraRS could be detectably labeled with Pra in the presence of Met in the growth medium as observed by in-gel fluorescence after click chemistry with Cy5-azide; analysis with MALDI-TOF mass spectrometry revealed that replacement of Met by Pra was approximately 10%.  NLL-MetRS and PraRS were used to cell-specifically label the respective proteomes of two bacterial strains co-cultured in media supplemented with Met, Anl, and Pra.  Treatment of the mixture of proteomes with a sequential copper-free and copper(I)-catalyzed click chemistry allows for precise fluorescent tagging of marker proteins based on their cellular origins.