Molecular cloning, expression, mRNA secondary structure and immunological characterization of mussel foot proteins (Mfps) (Mollusca: Bivalvia)

The macroscale production of mussel foot proteins (Mfps) in the expression system has not succeeded to date. The principal reasons for this are low levels of expression and yield of Mfps, lack of post-translational modifications (PTMs), and immunological toxic effects on the host system. Identification of post-translational modification sites, suitable expression hosts, and immunological responses through an experimental approach is very costly and time-consuming. However, in the present study, in silico post-translation modification, antigenicity, allergenicity, and the immunological reaction of all available Mfps were characterized. Furthermore, all Mfps were codon optimized in three different expression systems to determine the best expression host. Finally, we performed the in-silico cloning of all codon-optimized Mfps in a suitable host (E. coli K12, pET28a(+) vector) and analyzed the secondary structure of mRNA and its structural stability. Among the 78 Mfps, six fps are considered potential allergenic proteins, six fps are considered non-allergenic proteins, and all other fps are probably allergenic. High antigenicity was observed in bacterial cells as compared to yeast and tumor cells. Nevertheless, the predicted expression of Mfps in a bacterial host is higher than in other expression hosts. Important to note that all Mfps showed significant immunological activity in the human system, and we concluded that these antigenic, allergenic, and immunological properties are directly correlated with their amino acid composition. The study's major goal is to provide a comprehensive understanding of Mfps and aid in the future genetic engineering and expression of Mfps and its diverse applications in different fields.Communicated by Ramaswamy H. Sarma

Automated summary

The macroscopic production of mussel foot proteins (Mfps) in the expression system has not been successful due to low expression levels, lack of post-translational modifications (PTMs), and immunological toxicity. In this study, in silico characterization of PTMs, antigenicity, allergenicity, and immunological responses of Mfps were performed. Codon optimization was carried out in three expression systems, and the best expression host was determined. In-silico cloning of codon-optimized Mfps in a suitable host (E. coli K12, pET28a(+) vector) was performed, and the secondary structure and stability of mRNA were analyzed. Among the 78 Mfps, six are potential allergenic proteins, six are non-allergenic proteins, and the rest are likely allergenic. Bacterial cells showed higher antigenicity and predicted expression of Mfps compared to yeast and tumor cells. It was concluded that the immunological properties of Mfps are related to their amino acid composition. The study aims to provide a comprehensive understanding of Mfps and assist in future genetic engineering and expression of Mfps.