过度表达和净化来自大肠杆菌的有毒核酶
Erik J Daquilanea1, Meredith N Frazier2
1Department of Chemistry and Biochemistry, College of Charleston; erik.daquilanea@emory.edu.
Journal of visualized experiments : JoVE
|September 15, 2025
概括
在大肠杆菌中产生冠状病毒Nsp15是由于毒性而具有挑战性的. 使用C41(DE3) 细胞和优化的初始培养物显著提高了研究中这种必不可少的酶的产量.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 结构生物学 结构生物学
背景情况:
- 大肠杆菌 (E. coli) 是重组蛋白质生产的常见宿主.
- 毒性重组蛋白,如病毒核酶,可以抑制细胞生长并减少大肠杆菌的产量.
- 冠状病毒Nsp15内核酶表现出毒性,导致野生类型 (WT) 酶的产量较低.
研究的目的:
- 开发一种有效的方法来过度表达和净化E. coli中催化活性冠状病毒Nsp15的E. coli.
- 为了克服Nsp15在重组蛋白质生产过程中毒性所带来的挑战.
- 为了获得足够数量的纯化Nsp15用于生物化学和结构研究.
主要方法:
- 使用的C41(DE3) 大肠杆菌细胞,已知有毒蛋白质的增强表达.
- 在同一天使用单个殖民地初始培养物,以最大限度地减少滞后阶段和毒性.
- 表达Nsp15与一个N端6xHis标签用于净化.
- 使用基亲和染色学和尺寸排除染色学 (SEC) 净化Nsp15.
主要成果:
- 成功过度表达和净化野生型 (WT) 马冠状病毒Nsp15.
- 实现了足以用于下游生物化学分析的纯化Nsp15的产量.
- 获得的蛋白质适用于冷电子显微镜结构研究.
- 证明了优化的表达策略可以减轻NSP15的毒性.
结论:
- 结合C41(DE3) 细胞和优化起始培养,有效降低了Nsp15在大肠杆菌中的毒性.
- 这种方法可以产生高产量的活性Nsp15.
- 纯化的NSP15适合进行详细的生化和结构研究,推进冠状病毒研究.
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