title: The monotopic membrane protein human oxidosqualene cyclase is active as monomer.
abstract: The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.
authors.affiliation: F. Hoffmann-La Roche Ltd., Pharma Research Discovery, CH-4070 Basel, Switzerland.
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
authors.forename: Francis
authors.initials: F
authors.lastname: Müller
authors.forename: Brigitte
authors.initials: B
authors.lastname: D'Arcy
authors.forename: Martine
authors.initials: M
authors.lastname: Stihle
authors.forename: Eric
authors.initials: E
authors.lastname: Kusznir
authors.forename: Corinne
authors.initials: C
authors.lastname: Handschin
authors.forename: Olivier H
authors.initials: OH
authors.lastname: Morand
authors.forename: Ralf
authors.initials: R
authors.lastname: Thoma
mesh_terms: D001577:Benzophenones / Q000494:pharmacology; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology*; D002784:Cholesterol / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020128:Inhibitory Concentration 50; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000502:physiology*; D007810:Lanosterol / Q000737:chemistry; D008024:Ligands; D008862:Microsomes, Liver / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013185:Squalene / Q000031:analogs & derivatives* / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: C002821:2,3-oxidosqualene; D001577:Benzophenones; D003902:Detergents; D008024:Ligands; D011994:Recombinant Proteins; C105726:Ro 48-8071; D007810:Lanosterol; D013185:Squalene; D004247:DNA; D002784:Cholesterol; D019751:Intramolecular Transferases; C021924:lanosterol synthase