Evaluating Signal Peptide Efficiency for Extracellular Protein Secretion for mRNA Vaccine Design

mRNA vaccines have emerged as promising platforms for the prevention of infectious diseases and cancer treatment. The antigenic protein has a signal peptide added to the N-terminus for extracellular secretion. However, it remains unclear whether the optimization of signal peptides has been sufficiently compared and examined for antigen protein secretion and immunogenicity. This study investigated the effects of various signal peptides on the extracellular secretion of a model protein, NanoLuc luciferase (Nluc), in different cell lines. We compared the secretion efficiency of Nluc fused to artificial (#38 and #34) and natural signal peptides (cystatin S, lactotransferrin, and tissue plasminogen activator) in human embryonic kidney 293, C2C12, and HepG2 cells. Luciferase assays and Western blot analysis revealed that the cystatin S signal peptide consistently induced the highest secretion of Nluc among all cell types tested. Notably, the cystatin S signal peptide outperformed previously reported tissue plasminogen activator signal peptides in terms of secretion efficiency. Furthermore, we observed no correlation between Nluc secretion and mRNA expression levels for each signal peptide, suggesting that enhanced secretion was not attributable to increased transcription. Our findings highlight the potential of the cystatin S signal peptide in enhancing the extracellular secretion of antigenic proteins in mRNA vaccines by improving the efficiency of protein translation.