哺乳动物 Bestrophin 离子通道的表达与纯化
Summary
离子通道的纯化往往具有挑战性, 但一旦达到, 它就有可能允许对通道的功能和结构进行体外调查。在这里, 我们描述的步骤, 以表达和纯化哺乳动物 bestrophin 蛋白, 一个家庭的 Ca2 +激活 Cl 通道.
Abstract
人类基因组编码四 bestrophin paralogs, 即 BEST1、BEST2、BEST3 和 BEST4。BEST1, 编码的BEST1基因, 是一个 Ca2 +激活 Cl 通道 ( CaCC) 主要表现在视网膜色素上皮 (RPE)。BEST1 的生理和病理意义被强调的事实是, 在BEST1基因的200多个不同的突变已经基因链接到至少五视网膜退行性疾病的频谱, 如最佳 vitelliform黄斑营养不良 (最佳疾病)。因此, 了解 bestrophin 通道在单分子水平上的生物物理学具有重要的意义。然而, 获得纯化的哺乳动物离子通道往往是一项具有挑战性的任务。在这里, 我们报告了哺乳动物 bestrophin 蛋白的表达与 BacMam 杆状病毒基因转移系统和他们的亲和性和大小排斥色谱纯化的协议。纯化蛋白在随后的功能和结构分析中有潜在的应用前景, 如脂质双层和晶体学中的电生理记录。重要的是, 这条管道可以适应研究其他离子通道的功能和结构。
Introduction
Bestrophins 是一个通过从细菌到人类的不同物种而保存的离子通道的家族1。在人类中, BEST1基因, 位于染色体 11q12.3, 编码的膜蛋白 Bestrophin-1 (BEST1), 主要表现在基底膜的视网膜色素上皮 (RPE) 细胞的眼睛2,3 ,4。由585种氨基酸组成, 其中第一 ~ 350 在物种间高度保守, 含有其跨膜区域, BEST1 在人类1、5、6中起 CaCC 作用。此外, 鸡和肺炎克雷伯杆菌的 BEST1 同系物的作用为 homopentamers7,8, 表明整个进化过程中的保护水平很高。
在人类中, BEST1基因的200多个突变已经临床上与一组称为 bestrophinopathies1,9的视网膜变性疾病有关。报告了五项具体 bestrophinopathies, 包括最佳疾病、成人发病 vitelliform 营养不良、常染色体显性 vitreoretinochoroidopathy、常染色体隐性 bestrophinopathy 和视网膜色素变性3、4 ,10,11,12,13,14。这些疾病, 导致视力下降, 甚至失明, 目前无法治愈。为了发展治疗治疗和潜在的个性化医学, 重要的是要了解这些BEST1疾病引起的突变如何影响 BEST1 通道15的功能和结构。为了这些目的, 研究人员需要获得纯化的 bestrophin (野生类型和/或突变) 通道, 并进行体外分析5,8。
第一个关键步骤是哺乳动物细胞中较高物种的 bestrophin 通道的表达。由于 HEK293-F 细胞的杆状病毒转导 (BacMam 系统) 是 heterologously 表达膜蛋白16、17的一种强有力的方法, 该协议利用优化的 BacMam 向量 (pEG BacMam) 进行鲁棒表达。靶蛋白18, 在这种情况下是哺乳动物 bestrophin 同源。该载体用于各种膜蛋白的表达, 包括 G 蛋白偶联受体、核受体和其他离子通道18。还有证据表明, 所生产的蛋白质适合晶体学18。在 HEK293-F 细胞中具有较高的表达水平, 可以用色谱法纯化蛋白质;具体地说, 在 bestrophins 的情况下, 可以使用亲和性和大小排除色谱。
一旦该协议对 bestrophin 通道进行微调, 则可以通过平面脂质双层和 X 射线晶体学分别对纯化后的蛋白质进行分析, 以5、8为其功能和结构。总之, 这些技术为 bestrophins 和其他离子通道的功能和结构研究提供了强有力的管道。
Protocol
Representative Results
Discussion
该协议描述了一种有用的管道, 用于表达和纯化哺乳动物 bestrophin 离子通道, 用于未来的体外分析。虽然 FPLC 设备是需要的大小排除色谱, 注射器泵是足够的所有步骤的亲和层析, 包括捆绑, 洗涤和洗脱。当使用注射器泵来推动溶液 (在注射器中) 通过一列, 这是必不可少的垫弹簧一侧, 以避免推动气泡进入柱。如果蛋白质的纯度在亲和层析后已经足够的后续实验, 大小排除色谱可以省略, 以便 FP…
Divulgations
The authors have nothing to disclose.
Acknowledgements
该项目由 NIH 赠款 EY025290、GM127652 和罗切斯特大学开办资金资助。
Materials
| HEPES | Fisher Scientific | AC327265000 | |
| NaCl | Fisher Scientific | AC446212500 | |
| Glycerol | Fisher Scientific | G33-500 | |
| Imidazole | Fisher Scientific | AC301870010 | |
| MgCl2 | Fisher Scientific | AC197530010 | |
| TCEP | Fisher Scientific | AA4058704 | |
| Aprotinin | Fisher Scientific | AAJ63039MA | |
| Leupeptin | Fisher Scientific | AAJ61188MB | |
| Pepstatin A | Fisher Scientific | AAJ20037MB | |
| Phenylmethylsulfonyl fluoride | Fisher Scientific | AC215740050 | |
| DDM sol-grade | Anatrace | D310S | |
| DDM anagrade | Anatrace | D310 | |
| Sf-900 II SFM | ThermoFisher | 10902179 | |
| FreeStyle medium | ThermoFisher | 12338018 | |
| NanoDrop spectrophotometer | ThermoFisher | ND-2000 | |
| High pressure homogenizer | Avestin | Emulsiflex-C5 | |
| HisTrap column | GE | 17-5248-01 | |
| Superdex-200 column | GE | 28990944 | |
| AKTA Pure | GE | 29018224 | |
| Ultra-15 centrifugal filter units | Millipore | UFC910024 | |
| Ultra-4 centrifugal filter units | Millipore | UFC810024 | |
| Ultra-0.5 centrifugal filter units | Millipore | UFC505024 | |
| Optima XE-90 Ultracentrifuge | Beckman Coulter | A94471 | |
| Mini-PROTEAN Tetra Cell | Bio-Rad | 1658004 | |
| Mini-PROTEAN precast gel | Bio-Rad | 4561084 | |
| T100 Thermal Cycler | Bio-Rad | 1861096 | |
| PolyJet transfection reagent | SignaGen | SL100688 | |
| pEG BacMam vector | Obtained from the Gouaux lab at Vollum Institute |
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