Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations
| Autor: | Lucile Guyot, Sarah Mohammed-Bouteben, Lucie Hartmann, Lydia N. Caro, Renaud Wagner |
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| Přispěvatelé: | Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Detergent
[SDV.BIO]Life Sciences [q-bio]/Biotechnology Lipid particles reconstitution Extraction Biochemistry law.invention Pichia pastoris Membrane Lipids 03 medical and health sciences Recombinant expression Structural Biology law Integral membrane protein Purification 030304 developmental biology 0303 health sciences biology Protoplast Chemistry 030302 biochemistry & molecular biology Membrane Proteins Sciences du Vivant [q-bio]/Biotechnologies General Medicine biology.organism_classification Recombinant Proteins Yeast Membrane Membrane protein Solubilization Saccharomycetales Recombinant DNA Nanoparticles Integral membrane proteins |
| Zdroj: | Current protocols in protein science Current protocols in protein science, Wiley, 2020, 100 (1), pp.e104. ⟨10.1002/cpps.104⟩ |
| ISSN: | 1934-3655 |
| DOI: | 10.1002/cpps.104 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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