在大肠杆菌中生产和表征SARS-CoV-2 PLpro的协议
Anna De Falco1, Rebecca Greene-Cramer1, Ben A Shurina1
1Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
STAR protocols
|July 19, 2025
概括
本研究详细介绍了一种在大肠杆菌中产生SARS-CoV-2帕帕因类蛋白酶 (PLpro) 的方法. 该协议确保了用于COVID-19抗病毒研究的本地PLpro的良好产量.
科学领域:
- 生物化学 生化学
- 分子生物学分子生物学
- 病毒学 病毒学
背景情况:
- SARS-CoV-2 帕帕因样蛋白酶 (PLpro) 是开发抗病毒治疗COVID-19的关键目标.
- 有效的活性PLpro的生产对于药物发现和结构研究至关重要.
研究的目的:
- 建立一个强大的协议,用于重组表达和净化 SARS-CoV-2 PLpro 在大肠杆菌.
- 提供用于表征产生的PLpro和为NMR分析准备同位素丰富样本的方法.
主要方法:
- 在大肠杆菌中,PLpro作为SUMO融合蛋白的表达.
- 使用亲和染色学进行净化,然后对SUMO标签进行切割.
- 通过质谱学,NMR光谱学和酶活性试验进行表征.
- 为先进的NMR研究准备同位素丰富样本.
主要成果:
- 成功地生产了SARS-CoV-2 PLpro,产量很好.
- 演示方法,以获得酶在其原生,活性形式.
- 建立表征技术,包括质谱,核磁共振和酶分析.
结论:
- 本方案提供了一种可靠的方法,用于生产和表征SARS-CoV-2 PLpro.
- 这一资源将促进进一步研究针对PLpro.pl的COVID-19抗病毒药物开发.
相关概念视频
Conjugated Proteins
Simple proteins and protein complexes contain only amino acids. In contrast, many other proteins, called conjugated proteins, covalently bond with non-protein moieties.
Nucleoproteins are protein complexes that contain nucleic acids, categorized as deoxyribonucleoproteins (DNPs) or ribonucleoproteins (RNPs) respectively. The nucleosome is a typical example of a DNP where nuclear DNA is associated with histone proteins. The major antigen for the Covid-19 virus SARS-CoV is an RNP that is critical...
Nucleoproteins are protein complexes that contain nucleic acids, categorized as deoxyribonucleoproteins (DNPs) or ribonucleoproteins (RNPs) respectively. The nucleosome is a typical example of a DNP where nuclear DNA is associated with histone proteins. The major antigen for the Covid-19 virus SARS-CoV is an RNP that is critical...
Coronavirus
Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), are enveloped viruses characterized by their single-stranded, positive-sense RNA genome and helical nucleocapsid structure. The hallmark of these viruses is their club-shaped spike (S) glycoproteins that protrude from the viral envelope, facilitating attachment to host cells. Typically, coronaviruses infect the upper respiratory tract, often causing mild or asymptomatic disease. However, certain strains like...


