In Vitro Selection of Collagen‐Binding Vascular Endothelial Growth Factor Containing Genetically Encoded Mussel‐Inspired Adhesive Amino Acids

Protein immobilization technology is important in medical and industrial applications. We previously reported all‐in‐one in vitro selection, wherein a collagen‐binding vascular endothelial growth factor (CB‐VEGF) was identified from a fusion library of random and VEGF sequences. However, its interaction chemistry is mainly limited to the interaction established by the 20 canonical amino acids. Herein, we incorporated an adhesive non‐natural amino acid found in marine mussels, L‐3,4‐dihydroxyphenylalanine (DOPA), into the library for all‐in‐one in vitro selection. After selection, we identified DOPA‐containing CB‐VEGF. CB‐VEGF binds to collagen with an apparent dissociation constant of 2 nM; naïve VEGF does not bind to collagen. The collagen‐binding peptide domain of CB‐VEGF (CB‐peptide) exhibited stronger binding to collagen than a mutant peptide (substitution from DOPA to tyrosine), indicating the importance of DOPA to collagen binding. The collagen‐binding affinity of CB‐VEGF is 10‐fold higher than that of CB‐peptide, suggesting that the collagen‐binding ability of CB‐VEGF is not due to the additive function of CB‐peptide to VEGF, but is synergistic. Furthermore, increased cell growth was observed on CB‐VEGF‐treated collagen surfaces, not VEGF‐treated collagen surfaces. Thus, integrating all‐in‐one in vitro selection and DOPA incorporation shows promise in generating adhesive proteins on solid supports.