Reactivity of human antisera to codon optimized SARS-CoV2 viral proteins expressed in Escherichia coli

Abstract

Objective: The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV2 virus continues to pose a serious threat to public health worldwide. The development of rapid diagnostic kits can assist the Tzu Chi Foundation in supporting global volunteers working to provide relief during the current pandemic.

Materials and methods: In this study, nucleotide sequences derived from publicly available viral genome data for several domains of the SARS-CoV2 spike and nucleocapsid (N) proteins were chemically synthesized, with codon optimization for Escherichia coli protein expression. No actual viral particles were involved in these experiments. The synthesized sequences were cloned into an E. coli expression system based on pQE80L, and expressed viral proteins were subsequently purified using Ni-affinity chromatography. Western blotting was conducted using human antiviral sera to assess the response of codon-modified viral proteins to COVID-19 patient sera.

Results: N protein was expressed in amounts large enough to support large-scale production. The N-terminal domain, receptor-binding domain (RBD), Region 3, and the S2 domain were expressed in small but sufficient amounts for experiments. Immunoblotting results showed that anti-N IgG and anti-N IgM antibodies were detected in most patient sera, but only 60% of samples reacted with the recombinant RBD and S2 domain expressed by E. coli.

Conclusion: The results indicated that codon-optimized SARS-CoV2 viral proteins can be expressed in E. coli and purified for rapid antibody detection kit preparation, with the codon-optimized N protein, RBD, and S2 protein demonstrating the most potential.

Keywords: COVID-19; Codon optimization; Escherichia coli expression system; N protein; SARS-CoV2 antisera.

Figure 1

Schematic representation of the genome organization of SARS-CoV2 and functional domains of the S protein. (a) The single-strand RNA genome of SARS-CoV2 is mostly taken up by open reading frame 1a and open reading frame 1b, and the structural protein open reading frames that encode the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins are clustered near the 3' end. The structural proteins are common to all coronaviruses. (b) The S protein primarily consists of the S1 and S2 subunit (aa 671–1270). The S1 subunit can be further divided into an N-terminal domain (aa 27–297) and receptor binding domain (aa 310–583), and the latter includes the Region 3 domain (aa 391-525)

Figure 2

Agarose gel electrophoresis of the PCR products of cloned sequences. (a) N-terminal domain; (b) receptor binding domain; (c) S2 domain; (d) N protein; (e) R3 domain. (a-d) were analyzed on a 0.8% agarose gel, while (e) was analyzed on a 2% agarose gel. The size of PCR products of N-terminal domain, RBD, S2, N gene and R3 is 813 bp, 819 bp, 1,800 bp, 1,266 bp and 405 bp, respectively

Figure 3

Agarose gel electrophoresis of plasmid quick screening results and colony PCR results of R3 clones. (a) N-terminal domain, lane 11(clone 10); (b) receptor binding domain, lane 3 (clone 17), lane 7 (clone 21), and lane 11(clone 25); (c) S2 domain, lane 2 (clone 27), lane 5 (clone 30), lane 7 (clone 32), lane 10 (clone 35), lane 12 (clone 37), lane 13 (clone 38), and lane 16 (clone 41); (d) N protein; lane 3 (clone 47), lane 5 (clone 49), lane 11 (clone 55), lane 13 (clone 57), lane 14 (clone 58), and lane 15 (clone 59). (a-d) The recombinant plasmid contained possible inserted PCR products with lower mobility than the rest of lanes with empty pQE80 L plasmid. (e) R3 domain, PCR product analyzed on 2% agarose gel. Lane 4(clone 129), 6 (clone 164), 8 (clone 178), 10 (clone 187) contained PCR products with the same size indicated they are correct recombinant clones for R3 region. Lane 14 is PCR positive control

Figure 4

Protein expression and purification analysis by SDS-PAGE. (a) N-terminal domain (30.4 kDa), receptor-binding domain (30.6 kDa) and R3 (15.4 kDa) analysis by 15%SDS-PAGE. (b) S2 (66.6 kDa) analysis by 12% SDS-PAGE. (c) N protein (46.8 kDa) analysis by 12% SDS-PAGE. NI: non induction fraction, I: induction fraction, P: purified fraction. Arrows indicated the purified proteins

Figure 5

N protein response to anti-sera. (a) Anti-N IgG, (b) anti-N IgM detection by western blotting. Each lane contained 0.5 μg of purified N protein. Anti-sera in each lane was diluted 1000-fold. Secondary anti-human IgG or IgM was diluted 10,000-fold. Lanes 1–18 of each strip used different anti-sera, as listed in [Table 3]

Figure 6

Receptor-binding domain response to anti-sera. (a) Anti-receptor-binding domain IgG, (b) anti-receptor-binding domain IgM detection by western blotting. Each lane contained 1.5 μg of purified receptor-binding domain protein. Anti-sera in each lane was diluted 1000-fold. Secondary anti-human IgG or IgM was diluted 10,000-fold. Lanes 1-18 of each strip used different anti-sera, as listed in [Table 3]

Figure 7

S2 domain response to anti-sera. (a) Anti-S2 IgG, (b) anti-S2 IgM detection by western blotting. Each lane contained 8.5 μg of purified S2 domain. Anti-sera in each lane was diluted 1000-fold. Secondary anti-human IgG or IgM was diluted 10,000-fold. Lanes 1–18 of each strip used different anti-sera, as listed in [Table 3]