title: Overexpression of membrane proteins using Pichia pastoris.
abstract: Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.
authors.affiliation: Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, University of Strasbourg-CNRS, Illkirch, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bornert
authors.affiliation: Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, University of Strasbourg-CNRS, Illkirch, France.
authors.forename: Fatima
authors.initials: F
authors.lastname: Alkhalfioui
authors.affiliation: Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, University of Strasbourg-CNRS, Illkirch, France.
authors.forename: Christel
authors.initials: C
authors.lastname: Logez
authors.affiliation: Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, University of Strasbourg-CNRS, Illkirch, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Wagner
mesh_terms: D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D017408:Guidelines as Topic; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods* / Q000592:standards; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: C547713:ADRA1A protein, human; D008565:Membrane Proteins; D018340:Receptors, Adrenergic, alpha-1; D043562:Receptors, G-Protein-Coupled