DUAL BAIT TWO HYBRID SYSTEM

双饵二混合系统

• 批准号: 2713859
• 负责人: MARIJANE R RUSSELL
• 金额: $ 9.9万
• 依托单位: INVITROGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713859
• 关键词: chimeric proteins; genetic library; method development; protein purification; reagent /indicator; reporter genes; transfection /expression vector; yeast two hybrid system

DESCRIPTION (Adapted from the Investigator's Abstract): Biological regulatory systems require the specific organization of proteins into multi-component complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important concern using two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins, and the degree to which observed interactions are specific. This proposal outlines a strategy to create a novel dual bait system, designed to allow single-step screening of libraries for proteins which interact with protein 1 of interest fused to DNA-binding-domain A (LexA), but do not interact with protein 2 fused to DNA-binding-domain B (lambda cI). This will be a powerful technique to screen for false positives. The dual bait strategy also will allow for characterization of related family member proteins (wild type versus a mutated form of the same protein). This adaptation will add a second bait vector and a strain, which will have the appropriate auxotrophic markers for each bait to respond to for positive interactions. Each bait fusion protein will have a second unique reporter readout (GFP and beta-galactosidase) for positive protein/protein interactions. PROPOSED COMMERCIAL APPLICATION: Not available.

描述（改编自研究者摘要）：生物学 监管系统需要蛋白质的特定组织 多组分复合物。 两种混合动力系统已被用于 基于相互作用识别信号网络的新组件 与确定的伴侣蛋白。 使用两种混合动力的一个重要问题 系统是相互作用的蛋白质区分的程度 他们的生物伙伴来自进化上保守的相关蛋白质， 以及观察到的相互作用的具体程度。 这 提案概述了创建新型双诱饵系统的策略， 旨在允许对蛋白质库进行单步筛选， 与融合到 DNA 结合结构域 A 的感兴趣的蛋白质 1 相互作用 (LexA)，但不与融合到 DNA 结合域的蛋白质 2 相互作用 B（λCI）。 这将是筛选虚假信息的强大技术 积极的一面。 双诱饵策略也将允许表征 相关家族成员蛋白（野生型与突变型 相同的蛋白质）。 这种适应将添加第二个诱饵向量并且 一个菌株，每个菌株都有适当的营养缺陷标记 积极互动的诱饵。 各诱饵融合蛋白 将有第二个独特的报告读数（GFP 和 β-半乳糖苷酶） 用于积极的蛋白质/蛋白质相互作用。 拟议的商业应用：不可用。