PROTEIN INTERACTIONS: HIGH THROUGHPUT SUBCLONING

蛋白质相互作用：高通量亚克隆

• 批准号: 7602884
• 负责人: RUSSELL L FINLEY
• 金额: $ 13.96万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602884
• 关键词: Biological Assay; Cloning; Complex; Computer Retrieval of Information on Scientific Projects Database; Development; Escherichia coli; Funding; Genetic Recombination; Grant; In Vitro; Institution; Mass Spectrum Analysis; Methods; Open Reading Frames; Protein Microchips; Proteins; Proteomics; Research; Research Personnel; Resources; Source; System; United States National Institutes of Health; Yeasts; base; expression cloning; vector; yeast two hybrid system

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Further development and implementation of methods for high throughput (HT) subcloning. The ability to subclone many ORFs into different vectors is increasingly important to many proteomic analyses. Subcloning is essential, for example, for high throughput protein interaction assays including those based on the yeast two-hybrid system and protein microarrays. Subcloning is also necessary for determination of protein complexes by mass spectrometry, which requires expression of tagged proteins. It is also essential for structural determinations, which require purified proteins. We will further develop three approaches to high-throughput subcloning. One uses recombination in yeast, which is particularly well suited for constructing yeast expression clones for assays like the yeast two-hybrid system. The second approach uses recombination in E. coli, and the third uses a modified version of the GatewayTM in vitro cloning system developed by Invitrogen.

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构为 中心，但不一定是研究者所在的机构。 进一步开发和实施高通量（HT）亚克隆方法。 将许多ORF亚克隆到不同载体中的能力对于许多蛋白质组学分析越来越重要。 亚克隆是必不可少的，例如，高通量蛋白质相互作用的测定，包括那些基于酵母双杂交系统和蛋白质微阵列。 亚克隆对于通过质谱法测定蛋白质复合物也是必要的，这需要表达标记的蛋白质。 它对于需要纯化蛋白质的结构测定也是必不可少的。 我们将进一步开发三种高通量亚克隆方法。 一种方法是在酵母中进行重组，这特别适合于构建酵母表达克隆，用于酵母双杂交系统等试验。 第二种方法是利用E.第三种使用由Invitrogen开发的Gateway ™体外克隆系统的修改版本。