De Falco Anna, Greene-Cramer Rebecca, Shurina Ben A, Zakian Suren, Acton Thomas B, Ramelot Theresa A, Montelione Gaetano T
Center 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.
Center 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 Protoc. 2025 Jul 18;6(3):103952. doi: 10.1016/j.xpro.2025.103952.
Papain-like protease (PL) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. Here, we present a protocol for PL production in Escherichia coli. We describe steps for expressing PL as a fusion with a SUMO tag (small ubiquitin-like modifier) and purifying and obtaining it in its native form upon hydrolysis, with good yields. We provide approaches for PL characterization by mass spectrometry, NMR, and a fluorescence-based enzyme assay. We also detail a technique for producing isotope-enriched samples suitable for NMR studies.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的木瓜蛋白酶样蛋白酶（PL）是新冠病毒（COVID-19）抗病毒药物开发的重点靶点。在此，我们展示了一种在大肠杆菌中生产PL的方案。我们描述了将PL作为与小泛素样修饰物（SUMO标签）融合蛋白进行表达、纯化并在水解后以天然形式获得PL且产量良好的步骤。我们提供了通过质谱、核磁共振（NMR）和基于荧光的酶分析对PL进行表征的方法。我们还详细介绍了一种生产适用于NMR研究的同位素富集样品的技术。
