一种方便的自我清除的亲和标签方法,用于简单地净化无标签的重组蛋白
Sai Vivek Prabhala1, Sophia A Mayone1, Nathan M Moody1
1William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA.
Current protocols
|October 26, 2023
概括
一个新的自我切割的亲和度标签,iCapTagTM,可以单步净化无标签的标蛋白. 这种分体技术简化了蛋白质净化,加速了研究和功能研究.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 蛋白质净化 蛋白质净化
背景情况:
- 传统的蛋白质净化方法可能是复杂和耗时的.
- 亲和标签是常用的,但需要随后删除.
- 分裂内置技术为高效的蛋白质净化提供了一个潜在的解决方案.
研究的目的:
- 引入和验证一种用于蛋白质净化的新型自切割亲和度标签系统 (iCapTagTM).
- 为了证明iCapTagTM系统在单步净化过程中的效率和多功能性.
- 通过简化净化加速蛋白质表征和功能研究.
主要方法:
- 基于修改后的分裂蛋白 (Nostoc punctiforme DnaE) 的自切割亲和度标签的开发.
- 在捕获树脂上固定N端整体段的固定.
- 化C端整蛋白段与目标蛋白 (链酶,rHb1.1) 进行表达.
- 通过结合,洗和pH诱导的裂变 (pH8.5至6.2) 进行净化,以释放无标签的蛋白质.
- 展示基于柱式和无柱式净化格式.
主要成果:
- 成功单步净化高纯度,无标签的标蛋白 (链杆激酶和rHb1.1).
- 该iCapTagTM系统有效地捕获和释放高纯度的目标蛋白质.
- 在标准柱体净化和方便的无柱体格式中证明了适用性.
结论:
- 该iCapTagTM技术提供了一种高效和精简的方法,用于获得高度净化的无标签蛋白.
- 这种新型系统通过简化净化工作流程,显著加速下游应用.
- 通过蛋白质捕获科学,LLC商业化iCapTagTM,有助于在研究中更广泛地采用.
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