The human genome has opened a new chapter for drug discovery. In contrast to conventional drug discovery methods, which screen a drug library against a single protein target, the entire protein complement of the genome, i.e. the proteome, is now available for screening. A proteomic screen would greatly accelerate the drug discovery process by allowing potential interactions between a lead drug compound and an entire proteome to be determined. However, such drug-proteomic screens would be difficult to perform using conventional technology because of the need to rapidly convert genome to proteome, the requirement of engineering of proteins with specialized detection and affinity tags and the need for a platform that has the high throughput necessary to examine thousands of drug-protein interactions per hour. During Phase I, we have developed a new approach for drug discovery based on recent progress in the areas of in vitro protein expression, tRNA mediated protein engineering (TRAMPE) and photocleavable (PC) linkers such as PC-biotin. This technology allows the direct expression of individual proteins from genes or entire proteomes from genomes. A key to this technology is the incorporation of specially designed markers and affinity tags into proteins during their in vitro expression, thus facilitating their rapid isolation and detection. A central approach, termed PC-SNAG, allows proteins to be rapidly isolated from complex in vitro expression mixtures on beads and then photoreleased in pure form into solution or transferred directly onto the surface of a microarray substrate. During Phase II, proof-of-concept will be established for this new technology by creating a 321 member kinase library of in vitro expressed proteins (LIVE-PRO TM) that will be formatted on photoreleasable beads. In one assay, LIVE-PRO will be simultaneously screened to detect potential interactions with known kinase inhibitors. In a second assay, the effects of specific kinase inhibitors on the activity of each member of the LIVE-PRO will be simultaneously measured. This work will be performed in collaboration with Invitrogen, whose Gateway TM recombination technology and human Ultimate TM ORF Clones will allow creation of libraries of in vitro expressible plasmids. Dr. Mike Pavia, the former Chief Technology Officer at Millennium Pharmaceuticals and currently Entrepreneur-in-Residence at Oxford Bioscience will serve as a consultant.
| Lim, Mark; Rothschild, Kenneth J (2008) Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays. Anal Biochem 383:103-15 |
