Recombinant SARS-CoV-2 nucleocapsid protein: Expression, purification, and its biochemical characterization and utility in serological assay development to assess immunological responses to SARS-CoV-2 infection

Da Di, Mythili Dileepan, Shamim Ahmed, Yuying Liang, Hinh Ly

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay’s specificity. We expressed and purified from bacterial cells two recombinant forms of SARSCoV- 2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.

Original languageEnglish (US)
Article number1039
JournalPathogens
Volume10
Issue number8
DOIs
StatePublished - Aug 2021

Bibliographical note

Funding Information:
Funding: This research was supported in part by seed funds from the University of Minnesota Office of Academic Clinical Affairs’ COVID-19 Rapid Response Mechanism.

Funding Information:
This research was supported in part by seed funds from the University of Minnesota Office of Academic Clinical Affairs? COVID-19 Rapid Response Mechanism.

Publisher Copyright:
© 2021 by the authors.

Keywords

  • COVID-19
  • Diagnostics
  • ELISA
  • Nucleocapsid
  • RNA-binding protein
  • SARS-CoV-2

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