2024 Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins – A case study for Mefp-3 from Mytilus edulis

Authors:
Constanze Zwies^a, Angela María Vargas Rodríguez^a, Marcel Naumann^b, Franziska Seifert a,Markus Pietzsch^a

Journal:
Journal:  Protein Expression and Purification 219 (2024) 106483. DOI.org/10.1016/j.pep.2024.106483

Institute:

^a Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, Weinbergweg 22, 06120, Halle (Saale), Germany
^b Fraunhofer Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation, Weinbergweg 22, 06120, Halle (Saale), Germany


Abstract:

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role inadhesion. 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 and tag-free Mefp-3 using the SUMO-fusion technology.Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOFMS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.