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{"title":"毕赤酵母重组膜蛋白的制备及其分子研究","authors":"Lucile Guyot, Lucie Hartmann, Sarah Mohammed-Bouteben, Lydia Caro, Renaud Wagner","doi":"10.1002/cpps.104","DOIUrl":null,"url":null,"abstract":"<p><i>Pichia pastoris</i> 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.</p><p><b>Basic Protocol 1</b>: Production of membrane proteins in a <i>P. pastoris</i> recombinant clone using methanol induction</p><p><b>Basic Protocol 2</b>: Preparation of whole-membrane fractions</p><p><b>Alternate Protocol 1</b>: Preparation of yeast protoplasts</p><p><b>Basic Protocol 3</b>: Extraction of membrane proteins from whole-membrane fractions</p><p><b>Basic Protocol 4</b>: Purification of membrane proteins</p><p><b>Alternate Protocol 2</b>: Purification of membrane proteins from yeast protoplasts</p><p><b>Alternate Protocol 3</b>: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteins</p><p><b>Basic Protocol 5</b>: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.104","citationCount":"4","resultStr":"{\"title\":\"Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations\",\"authors\":\"Lucile Guyot, Lucie Hartmann, Sarah Mohammed-Bouteben, Lydia Caro, Renaud Wagner\",\"doi\":\"10.1002/cpps.104\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><i>Pichia pastoris</i> 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. 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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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