BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF PRION PROTEIN 2D CRYSTALS

朊病毒蛋白二维晶体的生物化学和生物物理表征

• 批准号: 8169775
• 负责人: STANLEY B PRUSINER
• 金额: $ 0.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169775
• 关键词: Benign; Biochemical; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Disease; Dose; Excision; Funding; Grant; Institution; Mass Spectrum Analysis; Molecular Conformation; Neurodegenerative Disorders; PrPSc Proteins; Preparation; Prions; Protein Isoforms; Proteins; Protocols documentation; Research; Research Personnel; Resources; Sampling; Scrapie; Source; Staining method; Stains; Structure; United States National Institutes of Health; base; particle; polyacrylamide gels; protein purification

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. Prions cause transmissible, neurodegenerative diseases with invariably fatal progression. A prion is an infective particle consisting exclusively of the prion protein. Two forms are discriminated: the benign, cellular form PrPC, and the disease-causing "scrapie" isoform PrPSc, which is confirmationally rearranged. Since PrPC and PrPSc differ in structure, studies of each conformation are essential. Our apporach focuses on PrPSc. We developed a high-yield protein purification protocol. Our preparations contain prion protein 2D crystals, which are structurally analyzed with standard and low-dose cryo electron microscopy. However, to further the quality and purity of our samples, we need to identify contaminants/copurified proteins from our samples for their systematic removal. This is the task for mass spectrometry. On a collaborative basis, copurified proteins are to be identified by analysis of bands derived from Coomassie-stained polyacrylamide gels.

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。 朊病毒会引起传染性神经退行性疾病，并且进展总是致命的。朊病毒是一种仅由朊病毒蛋白组成的感染性颗粒。区分两种形式：良性细胞形式 PrPC 和引起疾病的“痒病”亚型 PrPSc，后者已被确认重排。由于 PrPC 和 PrPSc 的结构不同，因此对每种构象的研究至关重要。我们的方法侧重于 PrPSc。我们开发了高产蛋白质纯化方案。我们的制剂含有朊病毒蛋白二维晶体，​​通过标准和低剂量冷冻电子显微镜对其进行结构分析。 然而，为了提高样品的质量和纯度，我们需要从样品中识别污染物/共纯化的蛋白质，以便系统地去除它们。这是质谱分析的任务。 在合作的基础上，通过分析考马斯染色聚丙烯酰胺凝胶的条带来鉴定共纯化的蛋白质。