In Vitro Selection of Collagen-Binding Vascular Endothelial Growth Factor Containing Genetically Encoded...

Area of Science:

• Biotechnology
• Protein Engineering
• Biomaterials Science

Background:

• Protein immobilization is crucial for medical and industrial applications.
• Previous methods identified collagen-binding vascular endothelial growth factor (CB-VEGF) but were limited by canonical amino acids.
• Marine mussel adhesive proteins inspired the incorporation of non-natural amino acids.

Purpose of the Study:

• To incorporate the adhesive non-natural amino acid L-3,4-dihydroxyphenylalanine (DOPA) into CB-VEGF for enhanced protein adhesion.
• To evaluate the collagen-binding properties and synergistic effects of DOPA-containing CB-VEGF.
• To assess the impact of CB-VEGF on cell growth on collagen surfaces.

Main Methods:

• All-in-one in vitro selection was employed to identify DOPA-containing CB-VEGF from a fusion library.
• Characterization of collagen-binding affinity using apparent dissociation constants.
• Comparative analysis of DOPA-containing peptides versus DOPA-mutated peptides (tyrosine substitution).
• Assessment of cell proliferation on collagen surfaces treated with CB-VEGF versus VEGF.

Main Results:

• A DOPA-containing CB-VEGF was successfully identified with strong collagen binding (apparent dissociation constant of 2 nM).
• The DOPA residue was critical for collagen binding, as evidenced by weaker binding in DOPA-mutated peptides.
• CB-VEGF demonstrated a synergistic collagen-binding effect, significantly stronger than the additive function of its components.
• CB-VEGF treatment of collagen surfaces promoted increased cell growth compared to untreated or VEGF-treated surfaces.

Conclusions:

• Integrating all-in-one in vitro selection with DOPA incorporation is a promising strategy for engineering adhesive proteins.
• DOPA-containing CB-VEGF exhibits enhanced collagen-binding affinity and promotes cell growth, indicating potential for biomaterial development.
• This approach offers a novel method for creating functionalized protein-based biomaterials for diverse applications.