A novel Mfp-4 derived fusion tag enhances recombinant mussel foot protein production and facilitates purification.

Abstract

Mussel foot proteins (Mfps) exhibit exceptional underwater adhesion and biocompatibility, rendering them highly valuable for biomedical and industrial applications. However, conventional extraction from natural sources suffers from low yields and high costs, limiting scalability. To address this challenge, we developed a dual-functional fusion tag derived from a 15-amino acid peptide segment of Mfp-4 (TM4) to enhance both expression and purification of Mytilus coruscus foot protein 5 (Mcofp-5). The fusion construct, TM4-Mcofp-5, demonstrated significantly improved soluble expression (0.5-0.6 g/L in shake-flask cultures), a ~3-fold increase compared to untagged Mcofp-5. Further optimization via fed-batch fermentation in a 7-L bioreactor boosted production to 2 g/L. Notably, TM4 enabled efficient single-step purification through nickel affinity chromatography, capitalizing on its intrinsic metal-chelating properties without requiring exogenous tags. Functional characterization revealed that TM4-Mcofp-5 retained superior adhesive strength in lap shear tests and enhanced cell proliferation, confirming bioactivity preservation. This strategy eliminates the need for non-native purification sequences, maintaining native protein functionality while significantly improving production efficiency. Our findings establish a scalable and cost-effective platform for high-yield Mfps production, with broad implications for tissue engineering, wound healing, and bioadhesive technologies.