Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease

Digant Nayak, Priscila dos Santos Bury, Marcin Drag, Alfred N. Nijkerk, Farid El Oualid, Shaun K. Olsen

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations


Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host’s key immune signaling proteins, thereby preventing the host’s innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Number of pages18
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Activity assay
  • Activity-based probes (ABPs)
  • COVID-19
  • DUB
  • Deubiquitinase
  • ISG15
  • PLpro
  • Papain-like protease
  • SARS-CoV-1
  • SARS-CoV-2
  • Ubiquitin
  • deISGylase

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology


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