In a multicellular animal like C. elegans, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. To address these challenges, we engineered a C. elegans phenylalanyl-tRNA synthetase (CePheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). We expressed the engineered CePheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells – and only those cells – to be labeled with azides upon feeding Azf-labeled E. coli to the worms. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affinity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins – including proteins with previously unknown expression patterns – expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; cell-specificity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we also developed a two-component system to drive overlapping – but not identical – patterns of expression of engineered CePheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-CePheRS system for highly efficient CePheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells.
Please note: Abstract shown here should NOT be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The Genetics Society of America
9650 Rockville Pike, Bethesda, MD
Phone: 301-634-7300, Fax: 301-634-7079
Questions and Comments: firstname.lastname@example.org