Expression,Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1) |
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| Authors: | Rajendra Boggavarapu Jean-Marc Jeckelmann Daniel Harder Philipp Schneider Z?hre Ucurum Matthias Hediger Dimitrios Fotiadis |
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| Institution: | Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.; Swiss Federal Institute of Technology Zurich, Switzerland, |
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| 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. |
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