Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis

• Published in: Analytical biochemistry 2023-02, Vol.662, p.114995-114995, Article 114995
• Main Authors: Sato, Ryo, Tomioka, Yui, Sakuma, Chiaki, Nakagawa, Masataka, Kurosawa, Yasunori, Shiba, Kohei, Arakawa, Tsutomu, Akuta, Teruo
• Format: Article
• Language: English
• Subjects: Agarose native; Arginine; Coronavirus Nucleocapsid Proteins - chemistry; Coronavirus Nucleocapsid Proteins - metabolism; COVID-19 - diagnosis; Electrophoresis - methods; Electrophoresis, Agar Gel - methods; Escherichia coli - genetics; Escherichia coli - metabolism; High level soluble expression; Humans; Nucleoprotein; Nucleoproteins; Recombinant Proteins - chemistry; SARS-CoV-2; SARS-CoV-2 - chemistry; SARS-CoV-2 - metabolism; Sepharose
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2022.114995

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle. [Display omitted] •Concentration-dependent structural changes of SARS-CoV-2 NP were detected by our newly developed agarose native gel electrophoresis.•The theoretical isoelectric point of NP indicates that it is a basic protein, but at high concentrations it behaves like an acidic protein.•Highly purified tag free recombinant protein should enable us to understand not only the characteristics of NP in solution but also its possible role in the viral life cycle.