The COVID-19 pandemic has had a wide range of implications at the medical, social, political, and financial levels (1–4). Although the introduction of the genetically engineered vaccine, mRNA vaccine, live adenovirus vector vaccine, and inactivated vaccine effectively controlled the SARS-CoV-2 pandemic and considerably reduced the severe morbidity and mortality associated with COVID-19, toward the end of 2020, there have been numerous mutations in the SARS-CoV-2 strains, of which three variants have been classified as variants of interest (VOI): EG.5 variant, XBB.1.5 mutant, and XBB.1.16 mutant (5–8). Owing to the enhancement of the immune escape ability of the mutants, the proportion of mutants in the global epidemic strains has rapidly increased, demonstrating a stronger transmission advantage (9, 10). The neutralizing ability of the neutralizing antibodies produced during previous infections decreased in some mutants, which has raised concerns regarding the “viral immune escape (11, 12).” Therefore, it is important to closely monitor future virus mutations and other types of transmission. There is an urgent requirement to develop universal and novel coronavirus vaccines and drugs.
SARS-CoV-2 is the seventh coronavirus that causes infection in humans in addition to low pathogenic members HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, as well as highly pathogenic SARS-CoV and MERS-CoV (13). The genome of SARS-CoV-2 is approximately 30,000 bases comprising two large overlapping open reading frames (ORF1a and ORF1b) and encodes four structural proteins, namely spike, envelope, membrane, and nucleocapsid (N) proteins, as well as nine contactable proteins (14). ORF1a and ORF1b undergo further processing to produce 16 nonstructural proteins (Nsp1–16) (13). Among the virus proteins, N protein is the core component of the virus (15). It is a heterostructure, 419 amino acid long, multidomain RNA binding protein. Similar to other novel coronavirus, SARS-CoV-2 N protein has two conserved, independently folded domains, called N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by an inherently disordered region (IDR) called the central linking region (LKR) (16). LKR includes a Ser/Arg (SR)-rich region that contains putative phosphorylation sites. In addition, there are usually two IDRs on both sides of the NTD and CTD of the coronavirus known as the N-arm and C-tail, respectively (16, 17). NTD, CTD, and IDR are responsible for RNA binding, RNA binding and dimerization, and regulating RNA binding activity and oligomerization of NTD and CTD, respectively (15, 18).
Among the novel coronavirus proteins, N protein is also the most abundant protein in the virion and is the decisive factor of virulence and pathogenesis (19). It binds to the viral genomic RNA and packages the RNA into a ribonucleoprotein complex (14, 15, 19). In addition to assembly, N proteins have other functions, including transcription and replication of viral mRNA and immune regulation (20). In particular, the N protein of SARS-CoV-2 has been found to counteract the host RNAi-mediated antiviral response through its double-stranded RNA binding activity as a viral inhibitor for RNA silencing, thus, making it a key target for diagnosis and vaccine and drug development (13, 19–21).
However, the basic function of N proteins is to bind genomic RNA and form protective nucleocapsids in mature viruses (14, 22, 23). The inherent ability of N protein to interact with nucleic acid makes its purification very challenging (22, 24). Moreover, N proteins can undergo liquid-liquid phase separation (LLPS) and promote the formation of dense liquid condensates, thereby increasing the difficulty of its purification (15, 25–27). Notably, there are significant differences in the structure and phase separation properties between nucleic acid contaminated and uncontaminated N proteins (24). Nucleic acid contamination may seriously affect the molecular properties of purified N protein, and thus, hamper the results of subsequent research and development of vaccines and drugs from N proteins. Polyethyleneimine (PEI) is a linear polymer, and the structural formula of its repeat unit is (-CH2CH2-NH-)n (28). The n value is generally 700–2,000, and the overall molecular weight of the polymer is 30–90 kDa. The presence of numerous repeated imine units makes PEI rich in positive charge in near-neutral solution, and thus, PEI can adsorb negatively charged biomolecules, such as nucleic acids or acidic proteins, form complexes, and initiate flocculation, resulting in the coprecipitation of PEI and bound biomolecules (28–31). Therefore, this study utilizes the characteristics of PEI binding to nucleic acids to remove nucleic acids from the N protein. This is followed by ammonium sulfate precipitation, dialysis, and nickel column purification to obtain the uncontaminated N protein.Thus, a simple process for the purification of full-length N protein without nucleic acid contamination was developed. At the same time, combined with virtual screening, inhibitors for nucleocapsid proteins were developed.