Genetic modification of bovine β-casein and its expression in the milk of transgenic mice

Genomic vectors containing mutant bovine β-casein with putative glycosylation sites were constructed to study the functional properties of glycosylated β-casein and its possible effects in milk. The mutation was performed by PCR-based site-directed mutagenesis. The tripeptide sequence, Asn-X-Ser, was generated between Asn₆₈ and Asn₇₃ in mature β-casein. The resulting β-casein mutants were designated pCJB68 and pCJB6873. pCJB68 carries a substitution of Ser₇₀ for Leu₇₀ (Asn₆₈-Ser₆₉-Ser₇₀-Pro₇₁), and pCJB6873 carries a substitution of Ser₇₀-Ser₇₁ for Leu₇₀-Pro₇₁ (Asn₆₈-Ser₆₉-Ser₇₀-Ser₇₁). The two mutated genomic constructs were placed under control of the bovine α-lactalbumin promoter, and lines of mice expressing the pCJB68 and pCJB6873 have been established. The milk from transgenic mice contained bovine β-casein at levels up to 2-3 mg/mL. N-Linked glycosylation of bovine β-casein in the pCJB6873 line was confirmed by peptide-N-glycosidase F treatment, but glycosylation of bovine β-casein did not occur in pCJB68 mice. In addition, mouse casein micelles containing glycosylated bovine β-casein showed the largest median diameter and rough outer surface, compared to normal mouse casein micelles and micelles from transgenic milk containing bovine β-casein.