FLUORESCENT PROTEIN TARGETS FOR CE-LIF DRUG SCREENING

More rapid and economical methods of drug +____________________________________ screening are sought by the pharmaceutical industry due to the dramatic projected increase in the number of "druggable" targets. This project aims to develop such a method based on the use of novel tRNA-based fluorescent labeling of the N-terminus of proteins combined with affinity capillary electrophoresis (ACE). AmberGen will evaluate a series of monofunctional and bifunctional detection/affinity tags, which will be specifically incorporated at the N-terminus of proteins during their cell-free expression using specially prepared tRNA conjugates. Because of the ability to uniformly fluorescence label target proteins while preserving their activity, potential drug interactions can be screened using ACE combined with light induced fluorescence detection (LIF). In this approach, small alterations in mobility can be detected using low volumes of reagents. Photocleavable affinity tags provide a means to rapidly isolate expressed proteins from the cell-free lysate. In addition, a series of "chemically addressable" moieties will be incorporated at the N-terminus of proteins for post-translational conjugation to molecules that cannot be accommodated by the native protein initiation system. In Phase II, an ACE-LIF drug screening technology based on cell-free N-terminal labeling will be optimized. Aclara Biosciences will assist in the transfer of this technology to microfluidic devices.