PROTEIN INTERACTIONS: HIGH THROUGHPUT SUBCLONING

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。高通量（HT）亚克隆方法的进一步发展和实施。将许多orf亚克隆成不同载体的能力对许多蛋白质组学分析越来越重要。例如，对于高通量蛋白质相互作用分析，包括基于酵母双杂交系统和蛋白质微阵列的分析，亚克隆是必不可少的。亚克隆对于质谱法测定蛋白质复合物也是必要的，这需要标记蛋白的表达。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。一种方法是在酵母中进行重组，这种方法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。第二种方法是在大肠杆菌中进行重组，第三种方法是使用由Invitrogen公司开发的改良版GatewayTM体外克隆系统。

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.