ANALYSIS OF THE YEAST PROTEOME USING PROTEIN CHIPS

使用蛋白质芯片分析酵母蛋白质组

• 批准号: 6782475
• 负责人: MICHAEL P. SNYDER
• 金额: $ 12.26万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6782475
• 关键词: Saccharomyces cerevisiae; binding proteins; chimeric proteins; fungal genetics; fungal proteins; galactose; genetic promoter element; glutathione transferase; guanine nucleotide binding protein; high throughput technology; method development; microorganism culture; open reading frames; phospholipids; phosphoprotein phosphatase; protein kinase

DESCRIPTION (Verbatim from Applicant's Abstract): 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.

描述（来自申请人摘要的逐字记录）：关于大多数 酿酒酵母基因，无论是新发现的还是以前发现的 识别，非常有限。目前，基因工程已成为研究的重点。 核酸水平的分析，如mRNA基因的研究 表达或基因组变异。然而，基因功能通常是在 水平的蛋白质分析，这在一般情况下没有受到高 吞吐量技术。我们建议开发高通量方法， 蛋白质芯片用于大规模的蛋白质功能生化分析， 酵母将制备一组表达酵母ORF作为GST的酵母菌株 在半乳糖诱导型启动子的控制下的融合;我们将 随后使用高通量方法进行大规模表达， 酵母蛋白蛋白质将被粘附在新型微孔芯片上， 正在发展并分析其生化活动。具体来说，我们将 使用体外结合和/或酶测定来鉴定GTP结合的靶点 蛋白质、磷脂结合蛋白和蛋白磷酸酶。我们还将 进一步分析蛋白激酶及其体外底物， 微芯片技术 我们希望这个项目能对科学产生重大影响。 社区生成的信息将保存在一个数据库中， 可以通过互联网访问。我们的质粒和酵母菌株 提供给所有感兴趣的研究人员。最重要的是， 预计将对酵母的研究产生巨大影响， 其他有机体。