TRNA LABELING OF CELLULAR EXPRESSED PROTEINS

The completion of the human genome project highlights a need for high-throughput analysis of gene expression at the protein level. Proteomics involves global analysis of the entire expressed complement of the genome. The general aim of this project is to introduce a powerful method for labeling nascent proteins produced in intact cells. This novel approach, termed TRACE-PRO (tRNA Labeling of Cellular Expressed Proteins) will open new avenues for studying cellular protein expression and protein-protein interactions. During Phase I, methods will be developed to efficiently introduce specially designed tRNAs into intact cultured mammalian cells. These tRNAs, which have already been developed and successfully evaluated in prokaryotic and eukaryotic cell-free lysates by AmberGen, act to introduce detection labels and affinity linkers into newly expressed proteins. Detection labels will be evaluated for their ability to replace more conventional radioactive labels to facilitate high throughput 2-D PAGE and capillary electrophoresis modes of detection and mass spectrometric identification of the labeled nascent proteins. Affinity linkers will be evaluated for their ability to facilitate identification of interacting cellular proteins. During Phase II, TRACE-PRO will be used to profile changes in protein expression and protein-protein interactions triggered in response to external stimuli such as addition of drugs. PROPOSED COMMERCIAL APPLICATION: The development of a cell-based protein expression labeling system using fluorescent and affinity tags will result in commercial products including reagents and hardware for proteomics research. AmberGen also plans to offer an in-house protein-protein interaction services which will be marketed to the pharmaceutical industry.