A fusion protein strategy for high-yield production of DOPA- modified recombinant mussel adhesive protein Pvfp-5P

Abstract

Mussel adhesive proteins (MAPs), particularly Pvfp-5 from Perna viridis, are promising bio-adhesives for biomedical applications, but their natural extraction is inefficient and recombinant production often suffers from low yield and insufficient DOPA modification. Here, we developed a fusion protein strategy to overcome these limitations. A chimeric gene encoding Pvfp-5β fused with a collagen-like preCol-P fragment (designated Pvfp-5P) was codon-optimized and expressed in Pichia pastoris GS115 together with a co-expressed tyrosinase from Bacillus megaterium for in situ conversion of tyrosine to 3,4 dihydroxyphenylalanine (DOPA). After 48 h of methanol induction in shake flasks, the recombinant protein was secreted at approximately 300 mg/L and purified to > 90% purity by single-step Ni-NTA affinity chromatography. The DOPA content reached 2.5 wt% determined by colorimetric assay.

Functional assays revealed that Pvfp-5P significantly promoted L929 fibroblast migration in a scratch wound healing assay, achieving 93.5% wound closure at 72 h (100 µg/mL), outperforming the natural MAP (83.5%). It also exhibited strong coating ability on both hydrophilic (glass) and moderately

hydrophobic (polystyrene) surfaces. Mechanical testing showed an extremely low elastic modulus of 2.34 kPa, indicating high flexibility and excellent interfacial conformability. This work provides a green, efficient, and scalable way for producing bioactive recombinant MAPs with promising applications in tissue repair, wound dressings, and surface engineering.

Keywords: Mussel adhesive protein, Pvfp-5, Pichia pastoris, DOPA, Tyrosinase.