Impact of different synonymous codon substitution strategies on SARS-CoV-2 nucleocapsid protein expression in Escherichia coli

Synonymous codon substitution, a gene engineering approach in synthetic biology, has been effective in improving the codon composition of recombinant genes of interest based on various criteria without altering the amino acid sequence. The SARS-CoV-2 virus nucleocapsid (N) protein is a stable, conserved and highly immunogenic that is less prone to mutation during infection, making it a key antigen in in vitro diagnosis, vaccine development, immunological and structural studies. While reports have focused on applying optimized N protein for different applications, the basic parameters used by different optimization tools for choosing the best approach for the N gene synonymous codon substitution are often neglected. Here, we analyzed the influence of different synonymous codon substitution strategies on SARS-CoV-2 N-protein expression in E. coli . Using different codon optimization (CO) and harmonization (CH) tools, we predicted and compared how parameters such as GC content, Codon Adaptation Index, codon quality and number of rare codons present in these sequences affect the N-protein expression. Our results also show that Minimum Free Energy (MFE) and RNA structure of N-term and C-tail of the N-protein coding sequence influence protein folding. We then predicted that the SR-rich region of the N-protein may contribute to slowing down the elongation rate during translation. This work presents a fundamental analysis of how different optimization tools affect SARS-CoV-2 N-protein expression and folding and suggests a basic approach to choosing the best strategy for optimal expression and folding of the protein for further studies. Graphical Abstract Highlights Codon substitution affects SARS-CoV-2 nucleocapsid (N) protein expression. SARS-CoV-2 N-protein expression varies with different codon optimization tools. RNA structures of N- and C-term impact RNA stability, protein expression and folding. SR-rich region of the N-protein may slow down elongation rate during translation.