Ruf Armin, Müller Francis, D'Arcy Brigitte, Stihle Martine, Kusznir Eric, Handschin Corinne, Morand Olivier H, Thoma Ralf
F. Hoffmann-La Roche Ltd., Pharma Research Discovery, CH-4070 Basel, Switzerland.
Biochem Biophys Res Commun. 2004 Mar 5;315(2):247-54. doi: 10.1016/j.bbrc.2004.01.052.
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.
单拓扑结构整合膜蛋白2,3-氧化角鲨烯环化酶（OSC）催化羊毛甾醇的形成，羊毛甾醇是哺乳动物中胆固醇的首个甾醇前体。因此，它是开发新型降胆固醇药物的重要靶点。在此，我们报道了功能性人OSC（hOSC）在毕赤酵母中的过表达及纯化。与来自人肝微粒体的OSC相比，重组hOSC对参考抑制剂Ro 48-8071的半数抑制浓度（IC50）几乎相同。分析超速离心数据与活性测量结果的相关性表明，单体hOSC具有最高的酶活性，这表明其为天然形式。此外，这些数据帮助我们确定了能使该蛋白成功结晶的去污剂。这种活性重组人膜蛋白的可得性是朝着更详细地对OSC进行功能和结构表征迈出的非常重要的一步。
