Constrained cell recognition peptides engineered into streptavidin

Streptavidin is widely used as an adaptor molecule in diagnostics, separati ons, and laboratory assay applications. We have here engineered cell adhesi ve peptides into the three-dimensional scaffolding of streptavidin to conve rt streptavidin into a functional protein. The mutations did not alter refo lding or tetramer assembly and the slow biotin dissociation rate of wild-ty pe streptavidin was retained. The peptide targets were hexapeptide sequence s derived from osteopontin and fibronectin that contain the RGD cell adhesi on sequence. Cell binding assays directly demonstrated that I at aortic end othelial cells and human melanoma cells adhered to surfaces coated with eit her of the two RGD streptavidin mutants in a dose-dependent fashion. Wildty pe streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alpha (v)beta(3) demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate t hat peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.