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.
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