PROTEIN INTERACTIONS: HIGH THROUGHPUT SUBCLONING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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许多蛋白质组学分析越来越重要。例如，亚克隆对于高通量蛋白质相互作用分析是必不可少的，包括基于酵母双杂交系统和蛋白质微阵列的分析。用质谱仪测定蛋白质复合体也需要亚克隆，这需要表达标记的蛋白质。它对于结构测定也是必不可少的，这需要纯化的蛋白质。我们将进一步开发三种高通量亚克隆方法。一种是在酵母中使用重组，这特别适合于构建酵母表达克隆，用于像酵母双杂交系统这样的分析。第二种方法使用大肠杆菌中的重组，第三种方法使用Invitgen开发的GatewayTM体外克隆系统的改进版本。

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.