From genes to proteins: High-throughput expression and purification of thehuman proteome

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. La wrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have p roduced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molec ular Analysis of Genomes and Their Expression) collection. This high-throug hput protein expression system has been developed for the analysis of the h uman proteome, the protein equivalent of the human genome, comprising the t ranslated products of all expressed genes. Functional and structural analys is of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of th is high-throughput expression system for protein production. A prototype wa s designed to demonstrate the feasibility of our approach. Using a PCR-base d strategy, 72 unique IMAGE cDNA clones have been used to create an array o f recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were per formed in 96-well microtiter plates, and are thus amenable to automation. E ach recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expr essed from this system are currently being analyzed for functional and bioc hemical properties, J. Cell. Biochem. 80:187-191, 2000. (C) 2000 Wiley-Liss , Inc.