Characterization and partial purification of bovine alpha-lactalbumin and beta-casein produced in milk of transgenic mice.

Bovine alpha-lactalbumin (alpha-LA) and bovine beta-casein (beta-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 alpha-IA and bovine beta-CN had molecular masses that were identical to those of those of the native proteins. The estimated expression of the proteins was 1.0 mg/ml of milk for alpha-LA and 3.0 mg/ml for beta-CN. The calcium binding of bovine alpha-LA suggested that the protein produced in murine milk has the same electrophoretic shift as native bovine alpha-LA after the removal of calcium. Nitrogen-linked glycosylation of native and murine synthesized bovine alpha-LA was identified by peptide-N-glycosidase F treatment, and the N-terminal amino acid sequence of HPLC-purified bovine alpha-LA from mouse milk was confirmed to be identical to native bovine alpha-LA. In addition, the phosphorylation of the bovine beta-CN expressed in the milk of transgenic mice was the same as that of native bovine beta-CN, as determined by phosphatase digestion.