Abstract
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
Highlights
Coronavirus disease 2019 (COVID-19) is caused by the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
We found that a concentration of 1 mg/ml of N122419 verified by Bradford assay and SDS-PAGE corresponds to A280 nm of 3.3
We obtained a suspension of N122-419-inclusion bodies (IB) that contained a high level of N, corresponding to 369 mg/L bacterial culture presenting a high purity, of more than 90%, as determined by analysis of the N122-419-IB band in SDS-PAGE (Fig 2A)
Summary
Coronavirus disease 2019 (COVID-19) is caused by the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The primary functions of SARS-CoV-2 N protein are binding to the viral RNA genome and packing them into a long helical nucleocapsid structure or ribonucleoprotein (RNP) complex. N protein binds to leader RNA, and maintains highly ordered RNA conformation, suitable for replicating and transcribing the viral genome [3]. This protein consists of three distinct domains: a N-terminal domain, NTD (46–174), that is responsible for RNA binding, an intrinsically disordered central Ser/Arg (SR)-rich linker (183–210) for primary phosphorylation and a C-terminal dimerization domain, CTD (258–361) responsible for oligomerization that is a RNA-binding domain. These charges are considered important for RNA binding, but they are potentially deterring for the self-association of the protein through electrostatic repulsion [4,5]
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