Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus foot protein-3 (Mfp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mfp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble Mfp-3s from the genus M. edulis (Mefp-3) and M. galloprovincialis (Mgfp-3) using the SUMO-fusion technology. 

Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of the peptide-bound tyrosines in Mfp-3s (containing ten tyrosine residues) for the first time. MALDI-TOF-MS confirmed that up to twelve hydroxyl groups were incorporated suggesting that not only DOPA but also 3,4,5-trihydroxyphenylalanine (TOPA) was formed. The highly hydroxylated Mfp-3s exhibited different adhesive properties. Modified Mefp-3 resulted in a tensile shear strength of 1.68 MPa, comparable adhesiveness to a commercial glue. These results demonstrate a concerted and simplified high yield production process of recombinant soluble Mfp3-based proteins with on demand DOPA modification. Mfp-3 and Mfp-3-based polymers modified with His₆-mTyr have the potential to become a practical bioadhesive for material science as well as for medical and biotechnological applications for future biomaterials.