DEVELOPMENT OF A FLOW CYTOMETRIC YEAST TWO HYBRID SCREENING SYSTEM

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。 为了在基因组水平上发现蛋白质相互作用组图，酵母双杂交筛选系统在过去的十年中得到了广泛的应用。尽管在适应液体处理机器人和阵列仪器方面付出了巨大的努力，但由于基于琼脂平板的营养标记选择和LacZ报告分析，目前的Y2H定位方法仍然是劳动密集型和低吞吐量的。我们最近开发的基于yEGFP的酵母双杂交系统使我们能够在没有平板的情况下用流式细胞术对Y2H检测培养物的GFP荧光进行定量分析。随着HyperCyt？高通量流式细胞仪采样平台的最新仪器进步，一盘384个Y2H液体培养样本可以在不到20分钟内进行定量分析。虽然这种系统非常适合于分析二元蛋白质-蛋白质相互作用对，但它没有足够的吞吐量来进行大的cDNA文库筛选，例如，对一个大的人类cDNA文库进行筛选可能需要几天的时间。为了解决这一限制，我们正在建立一种新型的sdY2H系统，该系统结合了表面显示报告程序作为额外的选择标记，允许通过简单的磁分离步骤快速丰富阳性细胞群。这种新的表面报告系统与以前建立的yEGFP报告系统一起，可以在液体培养中快速筛选cdna文库，为传统的基于平板的筛选和分析提供了一种高通量的替代方案。

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. To discover protein interactome maps at genomic scales, yeast two hybrid-based screening system has been used extensively in the past decade. Despite the tremendous effort in adapting liquid handling robot and array instrumentation, the current Y2H mapping approach is still labor intensive and low throughput attributed to the agar plating-based nutrient marker selection and LacZ reporter analysis. Our recent development of a yEGFP-based yeast two hybrid system enabled the quantitative analysis of GFP fluorescence of the Y2H assay cultures by flow cytometry without plating. With the recent instrumentation advancement of the HyperCyt¿ high-throughput flow cytometry sampling platform, a plate of 384 Y2H liquid culture samples can be quantitatively analyzed in less than 20 minutes. While such system is well-suited for analyzing binary protein-protein interaction pairs, it does not have adequate throughput to perform large cDNA library screening, e.g. sorting a large human cDNA library cells (typically ~109 cells) could take days. To address this limitation, we are establishing a novel sdY2H system that incorporates the surface display reporter as an additional selection marker allowing rapid enrichment of positive cell populations via a simple magnetic separation step. This new surface reporter together with the previously established yEGFP reporter system enable rapid cDNA library screening in liquid cultures, providing a high throughput alternative to conventional plating-based screening and analysis.