title: Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.
abstract: This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Boggavarapu
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hirschi
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Meury
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.affiliation: Division of Cellular and Molecular Neuroscience, Institut Universitaire en Santé Mentale de Québec, Québec, QC, Canada, G1J 2G3.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Bergeron
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland. dimitrios.fotiadis@ibmm.unibe.ch.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D008322:Mammals / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D018075:RNA, Complementary / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008565:Membrane Proteins; D018075:RNA, Complementary; D011994:Recombinant Proteins
keywords: 2D crystallization; Human; Mammalian; Membrane protein; Protein structure; Single particle analysis