Characterization and Partial Purification of Bovine α-Lactalbumin and β-Casein Produced in Milk of Transgenic Mice

Abstract Bovine α-lactalbumin (α-LA) and bovine β-casein (β-CN), from milk from transgenic mice were characterized and partially purified using electrophoretic, immunoblotting, and chromatographic methods. The transgenically expressed bovine milk proteins were identified using PAGE or by a combination of preparative isoelectrofocusing followed by Western immunoblotting. The heterologous bovine α-lA and bovine β-CN had molecular masses that were identical to those of the native proteins and pI values that were similar to those of the native proteins. The estimated expression of the proteins was 1.0 mg/ml of milk for α-LA and 3.0 mg/ml for β-CN. The calcium binding of bovine α-LA suggested that the protein produced in murine milk has the same electrophoretic shift as native bovine α-LA after the removal of calcium. Nitrogen-linked glycosylation of native and murine synthesized bovine α-LA was identified by peptide-N-glycosidase F treatment, and the N-terminal amino acid sequence of HPLC-purified bovine α-LA from mouse milk was confirmed to be identical to native bovine α-LA. In addition, the phosphorylation of the bovine β-CN expressed in the milk of transgenic mice was the same as that of native bovine β-CN, as determined by phosphatase digestion.

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