Nuclear Transcription Factor Translocation Assay

描述(申请人提供)：转录因子(Tf)是基因表达的关键调节因子。大多数转录因子存在于胞浆中，当被激活时，转位到细胞核以刺激基因活性。NFkappaB是一种具有重要治疗意义的转铁蛋白，它刺激细胞周期、增殖和炎症反应相关的基因，是抗癌和抗炎药物开发的重要分子靶点。由于核转位是NFkappaB调控基因表达能力的关键事件，测量NFkB转位的技术可以用于筛选阻止转位过程的新型抗肿瘤和抗炎药物。然而，很少有技术可以测量转录因子从胞浆到细胞核的运动，也没有一种技术可以用于高通量药物发现。DiscoveRx开发了一种酶片段互补(EFC)技术，可以使用极其敏感的β-半乳糖苷酶(β-GAL)互补试验来检测TF的易位。这种方法利用了两个基因工程的大肠杆菌β-半乳糖片段。较大的片段称为酶受体(EA)，在氨基端附近有缺失，而较小的片段称为酶供体(ED)，包含EA缺失的氨基末端序列。单独一家，艺电是不活跃的。然而，它可以与ED重新结合形成一种活性酶，该酶可以催化形成通过光度法检测为视觉放大反应的荧光产物。我们建议利用EFC技术建立一种直接检测NFkB易位的方法。我们将把ED改造成NFkB，并将EA定位到原子核。当ED-NFkappaB移位到细胞核时，它将能够与EA重新结合，生成活性B-Gal，该B-Gal可以催化形成荧光产物。作为测量NFkB易位的补充方法，我们将利用我们的EFC技术开发一种新的NFkappaB报告基因分析，其中NFkB反应元件驱动GST-ED融合蛋白的产生，该融合蛋白将在简化的基于细胞的荧光分析中用EA检测其表达。这些研究将开发用于药物发现的第一个NFkB易位。在未来，我们将把这项检测作为针对任何转铁蛋白的药物发现的通用技术，以开辟转录因子达哈拉科洛夫和治疗学的一个全新领域。

DESCRIPTION (provided by applicant): Transcription factors (TF) are key regulators of gene expression. Most TFs reside in the cytosol and when activated translocate to the nucleus to stimulate gene activity. A therapeutically important TF is NFkappaB, which stimulates genes involved in cell cycling, proliferation and inflammatory responses and is an important molecular target for the development of anti-cancer and anti-inflammatory drugs. Since nuclear translocation is a critical event in NFkappaB's ability to regulate gene expression, technologies that measure NFkB translocation could be used to screen for novel anti-neoplastic and anti-inflammatory agents that block the translocation process. However, there are few technologies to measure the movement of TFs from the cytosol to the nucleus and none that is amenable for high throughput drug discovery. DiscoveRx has developed an enzyme fragment complementation (EFC) technology that could be used to detect the translocation of TFs using an extremely sensitive beta-galactosidase (beta-gal) complementation assay. This method utilizes two genetically engineered fragments of E. coli beta-gal. The larger fragment, termed Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, termed Enzyme Donor (ED), contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, it can recombine with ED to form an active enzyme that can catalyze the formation of a fluorescent product detected photometrically as a visually amplified response. We propose to develop a direct assay for NFkB translocation using EFC technology. We will engineer ED into NFkB and target EA to the nucleus. When ED-NFkappaB translocates to the nucleus it will be able to recombine with EA to generate an active B-gal that can catalyze the formation of fluorescent products. As a complementary approach to measure NFkB translocation, we will develop a novel NFkappaB reporter gene assay using our EFC technology in which an NFkB response element drives the production of a GST-ED fusion protein whose expression will be detected with EA in a simplified cell based fluorescent assay. These studies will develop the first NFkB translocation for drug discovery. In the future, we will adapt this assay as a general technology for drug discovery against any TF to open up an entirely new field of transcription factor Dharmacoloav and therapeutics.