Kittredge Alec, Ward Nancy, Hopiavuori Austin, Zhang Yu, Yang Tingting
Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry.
Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry;
J Vis Exp. 2018 Aug 2(138):57832. doi: 10.3791/57832.
The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca-activated Cl channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.
人类基因组编码四种贝斯特rophin旁系同源物，即BEST1、BEST2、BEST3和BEST4。由BEST1基因编码的BEST1是一种主要在视网膜色素上皮（RPE）中表达的钙激活氯离子通道（CaCC）。BEST1基因中超过200种不同的突变已在基因上与至少五种视网膜退行性疾病相关联，如Best卵黄样黄斑营养不良（Best病），这突出了BEST1的生理和病理意义。因此，在单分子水平上了解贝斯特rophin通道的生物物理学具有重大意义。然而，获得纯化的哺乳动物离子通道通常是一项具有挑战性的任务。在这里，我们报告了一种使用杆状病毒BacMam基因转移系统表达哺乳动物贝斯特rophin蛋白并通过亲和色谱和尺寸排阻色谱进行纯化的方案。纯化后的蛋白质有潜力用于后续的功能和结构分析，如脂质双分子层中的电生理记录和晶体学。重要的是，该流程可用于研究其他离子通道的功能和结构。
