Purification and identification of osteopontin from bovine milk

Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A procedure for the isolation of OPN from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded ∼8.17 mg of purified protein (approximately 85% purity) per liter of bovine milk. The identity of the protein was verified by immuno-blotting. SDS-PAGE analysis revealed that the protein migrated at Mr 60,000. Thus, our procedure yielded biologically active OPN from an abundant and natural source.