High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae

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• 作者：Karen Molbaek (1)
  Peter Scharff-Poulsen (2)
  Claus Helix-Nielsen (3) (4) (5)
  Dan A Klaerke (1)
  Per Amstrup Pedersen (2)
  
  1. Department of Veterinary and Clinical Animal Science ; University of Copenhagen ; Dyrlaegevej 100 ; Frederiksberg ; DK-1870 ; Denmark
  2. Department of Biology ; University of Copenhagen ; Universitetsparken 13 ; Copenhagen OE ; DK- 2100 ; Denmark
  3. Department of Environmental Engineering ; Technical University of Denmark ; Miljoevej building 113 ; Kgs Lyngby ; 24105 ; Denmark
  4. Aquaporin A/S ; Ole Maaloesvej 3 ; Copenhagen N ; DK-2200 ; Denmark
  5. Laboratory for Water Biophysics and Membrane Technology ; University of Maribor ; Smetanova ulica 17 ; Maribor ; SL-2000 ; Slovenia
  
• 关键词：hERG ; Potassium channel ; Membrane protein production and purification ; Functional expression ; Yeast ; Cardiac action potential ; Drug screening ; Long QT ; Torsades de Pointes
• 刊名：Microbial Cell Factories
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：14
• 期：1
• 全文大小：2,915 KB
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• 刊物类别：Chemistry and Materials Science
• 刊物主题：Biotechnology
  Applied Microbiology
  Environmental Engineering/Biotechnology
  
• 出版者：BioMed Central
• ISSN：1475-2859

文摘

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.