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核细胞表达和净化hSox2-HMG域
Lijie Yang1,2, Wansen Tan1, Jingjun Hong1,2
1Institute of Health Sciences and Technology (IHST), Institutes of Physical Sciences and Information Technology, Anhui University, Hefei, China.
Bio-protocol
|August 28, 2025
概括
这项研究提出了一种高效的 prokaryotic 表达系统,用于生产高纯度的人类 Sox2 HMG (高流动性组) 域蛋白. 优化的方法确保了正确的折叠,并为未来的功能和结构研究提供了可扩展的策略.
科学领域:
- 分子生物学
- 蛋白质的表达和净化
- 基因调控
背景情况:
- 包括hSox2在内的Sox (与SRY相关的HMG盒) 蛋白家族对于细胞过程至关重要,但由于生产限制,其研究具有挑战性.
- 现有的Sox蛋白生产方法通常产生的量很少,折叠不当或净化效率低,阻碍了功能和结构分析.
研究的目的:
- 为人类 Sox2 HMG 域 (hSox2-HMG) 开发一种高效,具有成本效益和可扩展的 prokaryotic 表达系统.
- 获得高纯度,正确折叠的hSox2-HMG蛋白质,适用于结构和功能研究.
主要方法:
- 使用 pET22b 载体和 *Escherichia coli* BL21 ((DE3) 主体菌株构建了一个 prokaryotic 表达系统.
- 通过使用Ni-NTA亲和色谱,超过和尺寸排除色谱来优化IPTG诱导和净化蛋白质表达.
- 精制溶解和化条件以最大限度地提高蛋白质产量和纯度.
主要成果:
- 在大肠杆菌中成功实现了重组hSox2-HMG蛋白的高产量表达.
- 获得高纯度的hSox2-HMG蛋白质,其结构完整性和正确的折叠.
- 建立了一个高效,可扩展和具有成本效益的生产工作流程.
结论:
- 开发的原生体表达系统提供了生产功能性hSox2-HMG蛋白质的强有力的方法.
- 这种优化的协议有助于对hSox2-HMG的DNA结合和调节作用进行进一步的结构和功能研究.
- 这种易于获得的高纯度蛋白质有助于理解Sox2的生物功能.
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