毕赤酵母重组膜蛋白的制备及其分子研究

Q1 Biochemistry, Genetics and Molecular Biology

Current Protocols in Protein Science Pub Date : 2020-04-14 DOI:10.1002/cpps.104

Lucile Guyot, Lucie Hartmann, Sarah Mohammed-Bouteben, Lydia Caro, Renaud Wagner

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引用次数: 4

摘要

毕赤酵母是一种真核微生物，以其大规模生产重组蛋白的能力而闻名，包括整合膜蛋白，用于各种应用。这篇文章详细介绍了一系列的协议，朝着生产整体膜蛋白的进展，他们的提取和纯化在洗涤剂的存在，并最终在脂质纳米颗粒中重组。这些基本程序可以进一步优化，以提供完整的膜蛋白样品，这些样品与分子水平上的许多结构和/或功能研究兼容。每个协议提供一般指导方针，技术提示和具体建议，并与几个代表性哺乳动物蛋白质对应的案例研究说明。©2020 by John Wiley &基本方案1:利用甲醇诱导在pastoris重组克隆中生产膜蛋白基本方案2:制备全膜组分-替代方案1:制备酵母原生质体基本方案3:从全膜组分中提取膜蛋白基本方案4:纯化膜蛋白替代方案2:从酵母原生质体中纯化膜蛋白替代方案3:原生质体制备和膜增溶用于膜蛋白的纯化基本方案5:在脂质纳米颗粒中重建洗涤剂纯化的膜蛋白

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Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins, for various applications. This article details a series of protocols that progress towards the production of integral membrane proteins, their extraction and purification in the presence of detergents, and their eventual reconstitution in lipid nanoparticles. These basic procedures can be further optimized to provide integral membrane protein samples that are compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides general guidelines, technical hints, and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian proteins. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Production of membrane proteins in a P. pastoris recombinant clone using methanol induction

Basic Protocol 2: Preparation of whole-membrane fractions

Alternate Protocol 1: Preparation of yeast protoplasts

Basic Protocol 3: Extraction of membrane proteins from whole-membrane fractions

Basic Protocol 4: Purification of membrane proteins

Alternate Protocol 2: Purification of membrane proteins from yeast protoplasts

Alternate Protocol 3: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteins

Basic Protocol 5: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles

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来源期刊

Current Protocols in Protein Science Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： With the mapping of the human genome, more and more researchers are exploring protein structures and functions in living organisms. Current Protocols in Protein Science provides protein scientists, biochemists, molecular biologists, geneticists, and others with the first comprehensive suite of protocols for this growing field.