PDF(Pubmed)
Abstract：
Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.
摘要：
不同的发育信号和促死亡应激集中在细胞凋亡的线粒体途径的调节上。巴克斯，促凋亡的BCL-2效应子，直接在外部线粒体成员中形成蛋白脂质孔，以激活线粒体凋亡途径。BAX是各种人类疾病的可行药理靶点，并且已经做出了越来越多的努力来研究BAX的分子调控和识别选择性靶向BAX的小分子。然而,由于其易于聚集的性质，产生大量的单体和功能有能力的BAX一直具有挑战性。此外,尚不熟悉重组BAX生产的研究人员缺乏详细的指导性方案.这里,我们提出了一个全面的高产量表达方案，净化，并储存功能性重组BAX蛋白。我们利用内含素标记的BAX构建体，并采用两步色谱策略来捕获和纯化BAX，并提供观察BAX活化的标准测定实例。我们还强调了处理和存储BAX的最佳实践，以有效地保持其质量，保质期,和功能。
