Isolation and Biological Properties of Osteopontin from Bovine Milk

A procedure for the isolation of osteopontin (OPN) from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded ∼8 mg of purified protein per liter of milk. SDS–PAGE analysis revealed that the protein migrated atM_r60,000. NH₂-terminal sequence analysis of the first seven amino acids revealed the protein to be identical to that previously reported for bovine OPN. Also, our preparation demonstrated expected biological properties of OPN including adhesion of both endothelial and vascular smooth muscle cells to OPN in a dose- and Arg-Gly-Asp-dependent manner. Furthermore, OPN coupled to Sepharose was capable of binding the α_vβ₃integrin from a detergent extract of endothelial cells. Thus, our procedure yielded biologically active OPN from an abundant and natural source.