The monotopic membrane protein human oxidosqualene cyclase is active as monomer.
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.
Ruf
Armin
A
F. Hoffmann-La Roche Ltd., Pharma Research Discovery, CH-4070 Basel, Switzerland.
Müller
Francis
F
D'Arcy
Brigitte
B
Stihle
Martine
M
Kusznir
Eric
E
Handschin
Corinne
C
Morand
Olivier H
OH
Thoma
Ralf
R
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
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