Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations.
Fiche publication
Date publication
juin 2020
Journal
Current protocols in protein science
Auteurs
Membres identifiés du Cancéropôle Est :
Dr WAGNER Renaud
Tous les auteurs :
Guyot L, Hartmann L, Mohammed-Bouteben S, Caro L, Wagner R
Lien Pubmed
Résumé
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.
Mots clés
Pichia pastoris, detergent, extraction, integral membrane proteins, lipid particles reconstitution, protoplast, purification, recombinant expression
Référence
Curr Protoc Protein Sci. 2020 Jun;100(1):e104