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Membrane Composition Influences the Activity of in Vitro Refolded Human Vitamin K Epoxide Reductase

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.

Autoren:   Frank Jaenecke; Beatrice Friedrich-Epler; Christoph Parthier; Milton T. Stubbs
Journal:   Biochemistry
Jahrgang:   2015
DOI:   10.1021/acs.biochem.5b00716
Erscheinungsdatum:   09.10.2015
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