在列上的双梯度重新折叠,以有效地回收不溶性亲和标记重组蛋白质
Anna Vlaskina1, Dmitry Petrenko1,2, Yulia Agapova1
1National Research Center "Kurchatov Institute", Moscow, Russia.
Bio-protocol
|February 12, 2026
概括
这项研究引入了一种有效的方法,用于直接在Ni-TED树脂上重新折叠聚合重组蛋白,如CasV-M. 与传统技术相比,双梯度方法最大限度地减少了聚合,并提高了可溶性蛋白质的产量.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 蛋白质工程是指蛋白质工程.
背景情况:
- 在大肠杆菌中的重组蛋白质表达往往导致蛋白质聚合和纳入体的形成.
- 对于容易聚合的蛋白质,透析或稀释等传统的重新折叠方法可能是低效的.
研究的目的:
- 开发一种有效的协议,用于重新折叠聚合重组蛋白质,特别是那些形成纳入体的蛋白质.
- 提出一种新的双梯度重新折叠策略,用于Ni-TED树脂,作为传统方法的替代方案.
主要方法:
- 在Ni-TED树脂上直接使用双梯度重新折叠系统.
- 这种系统涉及散热剂度的逐步降低和非离子洗剂度的同时增加.
- 该协议使用CRISPR相关效应蛋白CasV-M的同类体来证明.
主要成果:
- 双梯度方法有效地减少了蛋白质聚合,并促进了原生蛋白质构成的恢复.
- 该方法表明,与透析或稀释相比,可再生性和可溶蛋白的产量有所改善.
- Ni-TED树脂促进了与高度β-甲醇的兼容性,并减少了非特异性结合.
结论:
- 开发的协议提供了一个有前途的策略,用于从包含体中获得可溶性,功能性重组蛋白质.
- 这种方法适用于广泛的聚合易发生的蛋白质,并为传统的重新折叠技术提供了替代方案.
- 在Ni-TED树脂上溶解,重新折叠和初始净化的集成工作流提高了效率.
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