A Streamlined Process to Phenotypically Profile Heterologous CDNAs in Parallel Using Yeast Cell-based Assays

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.