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.