全长重组朊蛋白的纯化与纤维化
Purification and Fibrillation of Full-Length Recombinant PrP.
作者信息
Makarava Natallia, Savtchenko Regina, Baskakov Ilia V
机构信息
Department of Anatomy and Neurobiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, 111 Penn St., Baltimore, MD, 21201, USA.
出版信息
Methods Mol Biol. 2017;1658:3-22. doi: 10.1007/978-1-4939-7244-9_1.
Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrP and methods for production of PrP de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrP for development of prion diagnostics including generation of PrP-specific antibody.
朊病毒蛋白的错误折叠和聚集与人类的几种神经退行性疾病有关，如克雅氏病、致死性家族性失眠症和格斯特曼-施特劳斯勒-谢inker病。朊病毒领域越来越多的应用，包括检测PrP的分析方法和从头生产PrP的方法，都需要高纯度和高质量的重组朊病毒蛋白（PrP）。在这里，我们报告了一种全长哺乳动物朊病毒蛋白表达和纯化的实验程序。该方案已被证明能产生极高纯度的PrP，且缺乏PrP加合物、氧化修饰或截断，这些通常是自发氧化或降解的结果。我们还描述了体外从重组PrP制备淀粉样纤维的方法。重组PrP纤维可以用作PrP的非感染性合成替代物，用于开发朊病毒诊断方法，包括产生PrP特异性抗体。
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