title: Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.
abstract: Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding and paves the way for the provision of pure, active membrane protein for structural studies.
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.|ZIK HALOmem , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Jaenecke
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Friedrich-Epler
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Parthier
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.|ZIK HALOmem , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014859:Warfarin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D014859:Warfarin; D064417:Vitamin K Epoxide Reductases