CHARACTERIZATION OF HOST-PATHOGEN PROTEIN-PROTEIN INTERACTIONS

宿主-病原体蛋白质-蛋白质相互作用的表征

• 批准号: 7957011
• 负责人: FRED L HEFFRON
• 金额: $ 3.25万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957011
• 关键词: Affinity; Arts; Biology; Biotinylation; Cellular biology; Computer Retrieval of Information on Scientific Projects Database; Dissection; Formaldehyde; Funding; Grant; Institution; Mammalian Cell; Mass Spectrum Analysis; Methodology; Methods; National Center for Research Resources; Proteins; Proteomics; Research; Research Personnel; Resources; Salmonella; Signal Transduction Pathway; Site; Source; United States National Institutes of Health; Virulence Factors; cell type; crosslink; link protein; pathogen; protein complex; protein protein interaction; vector

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. New mass spectrometry approaches have revolutionized cell biology by identifying the majority of proteins expressed in a given cell type. The methodology has been extended to the dissection of protein-protein interactions through affinity tags and purification of protein complexes. Using state of the art mass spectrometry we will identify cellular proteins that interact with Salmonella secreted virulence factors (so called type three secreted effectors; TTS effector). Specifically we will use a new tandem affinity tag together with new methods of protein-protein cross-linking to dissect the inflammasome and how one TTS effector inhibits NFK¿ activation via interaction with the Nod signal transduction pathway. We have constructed vectors containing a tandem affinity tag that are suitable for expression in mammalian cells as well as in Salmonella. The tag encodes two 6x His tags separated by a biotinylation site allowing purification of the tagged proteins under fully denaturing conditions. We will cross-link proteins with low concentrations of formaldehyde and attempt to use this tag to purify protein complexes using capabilities in the NCRR Center.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 新的质谱方法通过鉴定特定细胞类型中表达的大多数蛋白质，彻底改变了细胞生物学。 该方法已扩展到通过亲和标签和蛋白质复合物的纯化来剖析蛋白质-蛋白质相互作用。 使用最先进的质谱分析法，我们将鉴定与沙门氏菌分泌的毒力因子（所谓的三型分泌效应子；TTS 效应子）相互作用的细胞蛋白。 具体来说，我们将使用一种新的串联亲和标签以及蛋白质-蛋白质交联的新方法来剖析炎症小体，以及 TTS 效应器如何通过与 Nod 信号转导途径相互作用来抑制 NFK 激活。 我们构建了含有串联亲和标签的载体，适合在哺乳动物细胞以及沙门氏菌中表达。 该标签编码两个由生物素化位点分隔的 6x His 标签，允许在完全变性条件下纯化标记的蛋白质。 我们将用低浓度甲醛交联蛋白质，并尝试利用 NCRR 中心的功能使用此标签来纯化蛋白质复合物。