title: Expression, purification and low-resolution structure of human vitamin C transporter SVCT1 (SLC23A1).
abstract: Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Boggavarapu
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Jeckelmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.forename: Philipp
authors.initials: P
authors.lastname: Schneider
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.forename: Matthias
authors.initials: M
authors.lastname: Hediger
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D000818:Animals; D015870:Gene Expression; D006801:Humans; D009865:Oocytes / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060347:Sodium-Coupled Vitamin C Transporters / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D011994:Recombinant Proteins; C555162:SLC23A1 protein, human; D060347:Sodium-Coupled Vitamin C Transporters