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在大肠杆菌中表达未标记的arrestins及其净化

Sergey A Vishnivetskiy¹, Xuanzhi Zhan¹, Vsevolod V Gurevich¹

¹Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.

Current protocols

|September 6, 2023

概括

这项研究提出了一项强大的协议,用于从大肠杆菌表达和净化逮捕素 (细胞信号调节器). 该方法产生了高度纯净的,功能性的逮捕因,适合各种科学研究.

科学领域:

生物化学生物化学
分子生物学分子生物学
细胞信号传递细胞信号传递

背景情况:

阿雷斯蛋白是细胞信号通路的关键调节者.
生物化学,生物物理和结构研究需要纯化的逮捕因.
现有的净化方法对于某些类型或突变的阿雷斯可能是不够的.

研究的目的:

描述一种基本的,可扩展的协议,用于表达和净化无标签的野生类型和突变性逮捕因.
为下游研究提供一种可靠的方法来获得功能性逮捕因.
详细说明实现适用于敏感测试的高纯度逮捕因的步骤.

主要方法:

在大肠杆菌中逮捕因的表达.
硫酸从细胞溶解物沉.
使用氨酸-氨酸,Q-氨酸或SP-氨酸的序列色谱.
使用无约束柱来去除污染物.

主要成果:

成功大规模表达和净化无标签的逮捕素.
达到高纯度的逮捕素,其度高达10毫克/毫升.
净化阿斯因通过多个冷解周期表现出完全的功能和稳定性.
协议可适应不同类型和突变.

结论:

描述的协议提供了一种可靠和通用的方法来获得纯化的逮捕素.
纯化的逮捕因适用于各种生物化学,生物物理和结构研究.
这种方法有助于研究阿雷斯的功能和细胞信号的调节.

关键词:

在被逮捕后被捕.染色体学染色体学染色体学表达方式表达方式表达方式净化净化净化净化再组合剂的使用情况.

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