The objective of this project is to develop new technology for the rapid screening of proteins that are expressed in vitro and derived from specific genes linked to cancer. Markers and photo cleavable linkers will be incorporated into proteins during cell-free synthesis with specially prepared misaminoacylated tRNAs. The incorporation of highly fluorescent amino acids into nascent proteins can significantly decrease the time needed to detect and characterize such proteins compared with conventional radioactive methods and is amenable to high throughput automation. The incorporation of proprietary photo cleavable linkers into nascent proteins facilitates their rapid affinity-based purification and downstream physical and functional analysis by a variety of techniques including gel-shift assays, mass spectrometry and capillary electrophoresis. This approach will be evaluated during Phase I by incorporating a variety of fluorescent markers and photo cleavable linkers into four model proteins, by analyzing the properties of these modified proteins and by testing various methods for the isolation and detection of these proteins. This technology will be evaluated at the clinical level for genetic diagnosis of chain terminating mutations in the APC gene during Phase I, and in Phase II will be extended to detect a variety of genetic mutations linked to other cancer syndromes.
The development of rapid methods for the detection and isolation of cell- free produced proteins will have commercial applications in many areas of cancer research and clinical diagnostics. Products include reagents, kits for incorporating fluorescent markers and PC-linkers into nascent proteins, and automated systems for the screening of genetic defects such as the protein truncation test (PTT).
