title: Constrained cell recognition peptides engineered into streptavidin.
abstract: Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type 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 alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that 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.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
authors.forename: T C
authors.initials: TC
authors.lastname: McDevitt
authors.forename: K E
authors.initials: KE
authors.lastname: Nelson
authors.forename: P S
authors.initials: PS
authors.lastname: Stayton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D009842:Oligopeptides; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D019809:Streptavidin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D015815:Cell Adhesion Molecules; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D019809:Streptavidin