Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli

Abstract

The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

Keywords: ELISA; SARS-CoV-2; expression; nucleocapsid; recombinant protein; ribonucleoproteins.

Figure 1.. Structure of nucleocapsid protein.

The structure of Ncap is shown rendered from 8FD5 in two views rotated 180° around a central y axis. Helical regions are shown in cyan and β strands in magenta connected by loops possessing poorly-defined secondary structure. N and C terminal regions are labelled. Mutated residues in the proteins studied here are labelled and shown as spheres (carbon grey, oxygen red, nitrogen blue). The CASP6 cleavage site is shown in dark blue sticks (residues 399–402). Compared with the wild-type sequence, B.1.1 contains two amino acid variations, (Arg203Lys, and Gly204Arg), Alpha contains four (Asp3Leu, Arg203Lys, Gly204Arg and Ser235Phe), as does Delta (Asp63Gly, Arg203Met, Gly215Cys and Asp377Tyr) while Omicron has a deletion of Glu31-Ser33 (dark grey spheres) and two amino acid substitutions (Arg203Lys and Gly204Arg).

Figure 2.. Impact of Omicron variant mutations on Ncap structure.

Wild type Ncap structure (cyan cartoon, based on PDB entry 8FD5) was aligned with the predicted structure of the Omicron variant (magenta) as shown in the left panel. The two structures overlay well except in the region of the Omicron deletion. In Omicron, the deletion of residues 31–33 results in two adjacent glycine residues (residue 30, orange spheres and 31, previously 34 in the WT, magenta spheres). The side chains of residues Ala-Arg-Ser-Lys (35–38 in the WT sequence) show the largest structural changes (dotted yellow line show displacement in Å). Mutation of proline 13 to leucine (P/L 13) is predicted to have minimal structural impact.

Figure 3.. Interactions between the Ncap linker region and NSP3.

(A) The ensemble NMR structure of Ncap (cyan cartoon) in complex with NSP3 (PDB code 7PKU). Glycines 204 and 215 are shown as spheres, in cyan and orange respectively. Ser235 is labelled and shown as blue sticks and Arg203 shown as cyan sticks. (B) Ser235 approaches closest of all residues in the ensemble, coming to within 5 Å of NSP in one confirmation as shown. (C) A potential π-cation interaction between the Phe235 mutation present in the Ncap Alpha variant was modelled.

Figure 4.. Expression and purification of 6His-tagged SARS-CoV-2 Ncap Omicron.

SDS–PAGE (10%) analysis of samples from example purification. Molecular weight markers (MW, Bio-Rad (Cat. 161-0362)) are shown for each panel. Left panel shows total SDS-cell lysates of uninduced and induced E. coli BL21 carrying plasmid pT5P_MHTOmicronNcap (Lanes 1 and 2 respectively). The soluble fraction from induced cells after lysis, sonication and centrifugation (Lane 3). Pellet from 5% to 17% ammonium sulfate precipitation (Lane 4) and corresponding supernatant (Lane 5). The ammonium sulfate-protein pellet was resuspended in HisTrap loading buffer, centrifuged to separate into insoluble material (Lane 6) and the soluble fraction (Lane 7) and loaded on a HisTrap column (Lane 8). Lane 9 shows proteins passing through the HisTrap column. Pooled fractions eluted from the HisTrap column (Lane 10) were diluted with cation exchange loading buffer and loaded on SP Sepharose column (Lane 11). Flow through from SP column (Lane 12). Peak fractions from linear gradient elution (Lanes 13 and 14). Right panel shows samples of purified 6His-tagged; Ncap Omicron (Lane 1); Ncap Delta (Lane 2); Ncap Alpha (Lane 3); Ncap 203/204 (Lane 4); Ncap (Lane 5) as well as untagged Ncap (Lane 6).

Figure 5.. ELISA assay results for nucleocapsid variants with pooled SARS-CoV-2 positive human sera.

Ncap variants were immobilised on micro-titre plates and ELISA performed as described above (n = 2). Top left panel compares all Ncaps produced. Serum was diluted from 1/200 by eleven 1.75-fold serial dilutions. The original wild-type SARS-CoV-2 Ncap (untagged) and the his-tagged version are compared (top right). The remaining panels show pairwise comparisons between the latter and other 6His-tagged variants.

Figure 6.. Nucleocapsid proteins elicit weak response from pre-pandemic sera.

Ncap variants were immobilised on micro-titre plates and ELISA performed as described in Materials and Methods. All 32 serum samples testing positive for SARS-CoV-2 by PCR (PCR+) exhibited higher signal levels than the 94 pre-pandemic samples in these assays. Means and SD are indicated.