Effects of a Shift of the Signal Peptide Cleavage Site in Signal Peptide Variant on the Synthesis and Secretion of SARS-CoV-2 Spike Protein.

The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed to evaluate the effects of signal peptide on the secretion and release of SARS-CoV-2 spike protein. Therefore, we constructed a signal peptide deletion mutant and three signal peptide site-directed mutants. The (H) region and (C) region in the signal peptide of L5F-S13I mutant have changed significantly, compared with wild type, L5F and S13I. We demonstrated the effects of signal peptide on the secretion and synthesis of RBD protein, finding that mutation of S13 to I13 on the signal peptide is more conducive to the secretion of RBD protein, which was mainly due to the shift of the signal peptide cleavage site in the mutant S13I. Here, we not only investigated the structure of the N-terminal signal peptide of the SARS-CoV-2 spike protein but also considered possible secretory pathways. We suggest that the development of drugs that target the signal peptide of the SARS-CoV-2 spike protein may have potential to treat COVID-19 in the future.

Enhancing the Immunogenicity of RBD Protein Variants through Amino Acid E484 Mutation in SARS-CoV-2.

In the context of the COVID-19 pandemic, conducting antibody testing and vaccination is critical. In particular, the continued evolution of SARS-CoV-2 raises concerns about the effectiveness of vaccines currently in use and the activity of neutralizing antibodies. Here, we used the Escherichia coli expression system to obtain nine different SARS-CoV-2 RBD protein variants, including six single-point mutants, one double-point mutant, and two three-point mutants. Western blotting results show that nine mutants of the RBD protein had strong antigenic activity in vitro. The immunogenicity of all RBD proteins was detected in mice to screen for protein mutants with high immunogenicity. The results show that the mutants E484K, E484Q, K417T-E484K-N501Y, and K417N-E484K-N501Y, especially the former two, had better immunogenicity than the wild type. This suggests that site E484 has a significant impact on the function of the RBD protein. Our results demonstrate that recombinant RBD protein expressed in E. coli can be an effective tool for the development of antibody detection methods and vaccines.