Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 315, Issue 2, Pages 247-254Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2004.01.052
Keywords
OSC; cyclase; membrane protein; Pichia pastoris; detergent; analytical ultracentrifugation; protein crystallization; lanosterol synthase; cholesterol
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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 IC50 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. (C) 2004 Elsevier Inc. All rights reserved.
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