新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能

Controllable preparation of novel charged nanodisc and its binding with cytochrome P450

分别采用氮气吹干法和旋转蒸发法制备由磷脂和膜支架蛋白组成的电荷型纳米盘,用凝胶过滤色谱对其尺寸分级,分析了其性能,考察了其与肝微粒体细胞色素P450的结合能力。结果表明,纳米盘外观澄清透明,微观呈圆盘状,平均直径10nm,在p H 7.4下Zeta电位为-19.86 m V;肝微粒体破碎液与纳米盘能很好结合,CO差示光谱在450 nm出现明显吸收峰,细胞色素P450含量为0.10 nmol/mg,比活比未经纳米盘处理时提高13.0倍,较传统方法提升1.5倍,且操作时间由数日缩短至数小时。电荷型纳米盘在结合膜蛋白细胞色素P450的同时,活性保持良好,在膜蛋白研究领域极具应用潜力。

Membrane proteins are associated with the phospholipid bilayers as integral membrane and have multiple and important functions, such as responsible for carrying out import and export of molecules and communication with the surrounding environment. It has been up to 60% of all currently used drugs target for therapeutic purposes and always been the emphasis of research in the fields of biology, medicine and material science. Membrane proteins are experiencing the bottlenecks due to their low contents, strong hydrophobicity and difficulty in purification. The pretreatment method of membrane proteins is to solubilize the membrane for isolation the membrane proteins using large amounts of detergents firstly, and then purified by reconstitution to restore their functions. Novel charged nanodiscs with both membrane protein binding and protecting were prepared with phospholipid and membrane scaffold protein using both nitrogen blow-drying method and rotary evaporation, respectively in this work. The nanodiscs had clear and transparent appearance, and they were uniform and disc-shaped with an average particle size of 10 nm and a charge of-19.86 m V under p H 7.4, further size fractionation by gel filtration chromatography. By adjusting the preparation conditions, the properties of nanodiscs could be controlled well. The nanodiscs had a good binding to cytochrome P450 in liver microsome. The CO difference spectrum analysis results showed that the protein-nanodisc system exhibited a strong absorption peak at 450 nm. The content of cytochrome P450 was about 0.10 nmol/mg and the specific activity was improved 13.0 fold, 1.5 times higher than that of the traditional method, the operation time reduced from several days to several hours. It demonstrated that cytochrome P450 can be bound and protected simultaneously.