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

TLDR

The SARS‑CoV‑2 nucleocapsid protein binds viral RNA to form a ribonucleoprotein complex, is highly conserved and immunogenic, and is widely used in serological assays, though its RNA‑binding propensity may compromise assay specificity. The study aims to develop methods for expressing, purifying, and characterizing SARS‑CoV‑2 nucleocapsid protein and to apply it in serological assays for detecting infection. We expressed and purified two recombinant N forms from bacterial cells—one RNA‑bound from the soluble fraction and one RNA‑free—and characterized their biochemical properties. Both recombinant N forms can be used in ELISAs to detect anti‑N antibodies and seropositive sera, with the RNA‑free form showing slightly higher sensitivity to mouse mAb, and EMSA revealed N preferentially binds ssRNA in a sequence‑independent manner via both NTD and CTD.

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 SARS-CoV-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.