Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis

Abstract

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

Keywords: COVID-19; Heterologous protein production; Immunological test; Structural characterization.

Fig. 1

r2N protein characterization. (A) Densitometry analysis of r2N protein by 7–18% gradient SDS-PAGE (40 μg of protein/lane) using Quantity One 1-D Analysis Software. (B) Western-blotting of the r2N protein using anti-His monoclonal antibody produced (1:3000) and anti-SARS-COV2 N protein polyclonal antibody (1:1000) and revealed with secondary antibodies conjugated to alkaline phosphatase (AP) and AP conjugate substrate kit. (C) IEF-PAGE in IEF phast gel pH 3–9. D. Chromatographic profile on Superdex 200 10/300 GL at 220 nm.

Fig. 2

Oligomeric state analysis of the r2N protein by EGS crosslinking assay evaluated at NUPAGE 4–12% Bis-Tris gel. (A) Crosslinking assay of the r2N protein produced by protocol I. Reactions were prepared with 10 μM of r2N protein in EGS concentrations ranging 0–5 mM. (B) Evaluation of E. coli nucleic acid contamination at the purified r2N protein produced by protocol I in SDS-treated or untreated samples by 1% agarose gel electrophoresis stained with GelRed. (C) The effect of buffer ionic strength upon oligomeric state was also evaluate. Reactions were prepared with 10 μM of r2N protein, in a fixed concentration of EGS (4 mM), ranging salt concentration 0.12–1.5 M NaCl. (D) The r2N protein purified by protocol II treated or untreated with RNAse A was also evaluated by crosslinking assay and by western blotting (E) using anti-SARS-COV2 N protein polyclonal antibody (1:1000). The mass of 1000 μg of r2N protein was treated using different ratios of RNAse A: r2N protein, (A) 1:15, (B) 1:5, (C) 1:1, (D) 5:1 and (E) 10:1. Asterisks highlight the RNAse A monomer and its multimers.

Fig. 3

Structural characterization of r2N protein. (A) Thermal denaturation in CD analysis monitored at Θ222 nm; (B) Intrinsic tryptophan fluorescence Ratio (F330/F350); (C) Spectral Center of Mass and (D) Light Scattering Area. r2N protein produced by protocol I in black line and by protocol II in white line.

Fig. 4

Distribution of optical density values from positive and negative samples used for r2N protein evaluation on the ELISA platform.