RESUMO
To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.
Assuntos
Proteínas Fúngicas/metabolismo , Proteoma , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucose/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Biblioteca de Peptídeos , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , Estreptavidina/metabolismoRESUMO
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
