Genetic Modification of Bovine β-Casein and Its Expression in the Milk of Transgenic Mice
Published in Journal of Agricultural and Food Chemistry, Volume 44, Issue 3, March 1, 1996, pages 953-960.
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.