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

Abstract

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

Keywords:

• Mussel foot proteins
• in silico analysis
• immunogenicity
• cloning
• mRNA structure

Acknowledgements

We thank the Department of Zoology, University of Calicut for providing the infrastructural facility.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Correspondence and request for materials should be addresses to PPA or YSV

Author contributions

Conceived, designed and carried out the experiments: P.P.A; Analyzation and interpretation of the data: P.P.A and Y.S.V; Wrote the main manuscript: P.P.A; Photographic plate preparation: Y.S.V; Supervision: Y.S.V; All authors reviewed the manuscript.

Data availability statement

The datasets generated and/or analyzed during the current study are presented in the main manuscript. Supplementary files are available in online repository Dryad (https://datadryad.org/stash/share/wrvvQ2kJExfJcW3rWW6q3LdzVWg0j3QeN9ZjSodB9Q0) .

Database used in the study

All data and in-silico tools used in the study are publicly available without any restrictions anybody can access. The accession number of each Mfps proteins are presented in Table 1. All methods were carried out in accordance with relevant guidelines and regulations.

Additional information

Funding

The present study financially supported by DST-SERB (EEQ/2021/000379). The first author thankful to the MOTA, Government of India for the financial assistance in the form of NFST.