Information about most Saccharomyces cerevisiae genes, whether newly discovered or previously identified, is very limited. Currently, much emphasis has been placed on gene analysis at the level of nucleic acids such as the study of mRNA gene expression or genome variation. However, gene function is often deduced at the level of protein analysis, which in general has not been subjected to high throughput techniques. We propose to develop high throughput methods and protein chips for the large-scale biochemical analysis of protein function in yeast. A set of yeast strains will be prepared that express yeast ORFs as GST fusions under the control of a galactose-inducible promoter; we will subsequently use high throughput methods for the large-scale expression of the yeast proteins. The proteins will be adhered to novel microwell chips that we are developing and analyzed for biochemical activities. Specifically, we will use in vitro binding and/or enzymatic assays to identify targets of GTP-binding proteins, phospholipid binding protein and protein phosphatases. We will also further analyze protein kinases and their in vitro substrates using protein microchip techniques. We expect this project to have a significant impact on the scientific community. The information generated will be maintained in a database that is accessible over the Internet. Our plasmids and yeast strains will be made available to all interested researchers. Most importantly the technology developed is expected to have an enormous impact on the study of yeast and other organisms.
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