A rapid method for purifying osteopontin from bovine milk and interaction between osteopontin and other milk proteins

Osteopontin (OPN) was isolated from bovine milk whey using a two-step chromatographic procedure. Acid whey was batch-processed with DEAE-Sephacel at pH 5.0, and an OPN-rich fraction (approximately 85% purity) was obtained from the first chromatographic steps. Subsequent POROS® HQ anion exchange HPLC yielded approximately 11 mg of purified OPN from 1 L of whey. The identity of the protein was verified by immuno-blotting and N-terminal amino acid analysis. To assess the function of OPN in milk, the milk proteins interacting with OPN were isolated by an immobilized OPN column. Lactoferrin and lactoperoxidase electrostatically bound to OPN. IgM had high affinity to OPN with KD=1.77×10-7M. Considering that these three proteins are also involved in the biophylactic system, it can be presumed that they are transported by OPN to their effector site and function either independently or in collaboration with OPN.