title: High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae.
abstract: The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His 8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His 8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His 8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His 8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays.
authors: Department of Veterinary and Clinical Animal Science, University of Copenhagen, Dyrlaegevej 100, Frederiksberg, DK-1870, Denmark. kmolbaek@sund.ku.dk.
authors: Karen
authors: K
authors: Molbaek
authors: Department of Biology, University of Copenhagen, Universitetsparken 13, Copenhagen OE, DK- 2100, Denmark. pspoulsen@bio.ku.dk.
authors: Peter
authors: P
authors: Scharff-Poulsen
authors: Department of Environmental Engineering, Technical University of Denmark, Miljoevej building 113, Kgs Lyngby, 24105, Denmark. Claus.Nielsen@fysik.dtu.dk.|Aquaporin A/S, Ole Maaloesvej 3, Copenhagen N, DK-2200, Denmark. Claus.Nielsen@fysik.dtu.dk.|Laboratory for Water Biophysics and Membrane Technology, University of Maribor, Smetanova ulica 17, Maribor, SL-2000, Slovenia. Claus.Nielsen@fysik.dtu.dk.
authors: Claus
authors: C
authors: Helix-Nielsen
authors: Department of Veterinary and Clinical Animal Science, University of Copenhagen, Dyrlaegevej 100, Frederiksberg, DK-1870, Denmark. dk@sund.ku.dk.
authors: Dan A
authors: DA
authors: Klaerke
authors: Department of Biology, University of Copenhagen, Universitetsparken 13, Copenhagen OE, DK- 2100, Denmark. papedersen@bio.ku.dk.
authors: Per Amstrup
authors: PA
authors: Pedersen
mesh_terms: D016589:Astemizole / Q000378:metabolism*; D018533:Biomass; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D039563:Histamine H1 Antagonists, Non-Sedating / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D039563:Histamine H1 Antagonists, Non-Sedating; C000606913:KCNH2 protein, human; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016589:Astemizole
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