title: Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.
abstract: Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research TransCure, University of Bern, Bern, Switzerland.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Bergeron
authors.forename: Rajendra
authors.initials: R
authors.lastname: Boggavarapu
authors.forename: Marcel
authors.initials: M
authors.lastname: Meury
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.forename: Luc
authors.initials: L
authors.lastname: Caron
authors.forename: Paul
authors.initials: P
authors.lastname: Isenring
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Hediger
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D000818:Animals; D051398:Aquaporin 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003460:Crystallization; D004530:Egg Yolk / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016624:Negative Staining; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D027981:Symporters / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D014982:Xenopus laevis / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D051398:Aquaporin 1; D011994:Recombinant Proteins; D027981:Symporters; C496020:AQP1 protein, human; C119651:SLC12A7 protein, human; D000096922:K Cl- Cotransporters