将SARS-CoV-2主蛋白酶捕获到短暂的人工生菌因子中,以实现高产量的表达和简化净化
Pavel Novotný1, Adéla Moravcová2, Veronika Nováková3
1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.
International journal of biological macromolecules
|February 13, 2026
概括
三种表达策略产生高质量的SARS-CoV-2主要蛋白酶 (Mpro) 用于研究. 所有方法都产生具有真实N-终端的活性Mpro,适合生物化学和结构研究.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 病毒学 病毒学
背景情况:
- SARS-CoV-2 复制依赖于主要蛋白酶 (Mpro) 来处理聚蛋白质.
- 活跃的Mpro需要一个真正的N端才能完全发挥酶功能.
- 高质量的Mpro对于生化,结构和抗病毒研究至关重要.
研究的目的:
- 为了比较三个不同的载体设计策略来表达活跃的SARS-CoV-2 Mpro.
- 评估获得具有真实N端的Mpro以获得最佳活动的方法.
- 为了确定表达的Mpro对于生物物理和结晶研究的适用性.
主要方法:
- 自动处理融合:SUMO-Mpro-HisTag构造具有自动催化释放的本地裂解部位.
- 外部蛋白酶裂变:需要ULP-1蛋白酶的HisTag-SUMO-Mpro融合.
- 自行处理的变种:HisTag-SUMO通过突变的裂变部位与Mpro联系在一起,以简化净化.
主要成果:
- 这三种策略都成功地产生了具有真实N-termini的Mpro.
- 表达的Mpro表现出可比的N端子,循环二极化谱,动力参数和热稳定性.
- 由此产生的Mpro蛋白质适用于生物物理分析和结晶.
结论:
- 多种表达策略可以产生适合研究的活跃SARS-CoV-2 Mpro.
- 选择表达策略可以适应其他对N端修饰敏感的蛋白酶.
- 这些方法有助于生产高质量的Mpro,用于关键的生化和结构研究.
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