Chemical and physical properties of bovine alpha-lactalbumin, beta-casein and genetically altered bovine beta-casein produced in the milk of transgenic mice

The genomic bovine $\alpha$-lactalbumin gene (1.7 kb) and the bovine $\beta$-casein gene (8.5 kb) linked to bovine $\alpha$-lactalbumin 5$\prime$ flanking region (2.0 kb) were expressed in two lines of transgenic mice, $\alpha$-114 and $\alpha\beta$ 34, respectively. Whey proteins, caseins, or skim milk proteins of the transgenic mouse milk containing bovine $\alpha$-lactalbumin or bovine $\beta$-casein were analyzed and separated by polyacrylamide gel electrophoresis (PAGE), using SDS, urea, or native (non-denaturing) conditions. Using PAGE followed by immunoblotting, both heterologous proteins were proved as identical as the native proteins in the molecular weight and the charge. The estimated expression level of bovine $\alpha$-lactalbumin and $\beta$-casein was about 1.0 mg/ml and 3.0 mg/ml, respectively. The expression level of bovine $\alpha$-lactalbumin was lactation-dependent, which was higher in early lactation than that in late lactation stage. Calcium binding of the heterologous bovine $\alpha$-lactalbumin was also the same as native bovine $\alpha$-lactalbumin. The phosphorylation level of the bovine $\beta$-casein expressed in the transgenic mouse milk was identical to the native bovine $\beta$-casein. The glycosylated bovine $\alpha$-lactalbumin and the N-terminal amino acid sequence of purified bovine $\alpha$-lactalbumin were characterized and determined. The physical properties of transgenic mouse milks were changed due to the heterologous expression of the bovine $\beta$-casein or $\alpha$-lactalbumin in the mouse milks. An increased moisture of transgenic mouse milk containing bovine $\alpha$-lactalbumin was observed. However, the milk composition of transgenic mouse milk containing bovine $\beta$-casein did not change significantly. In addition, two chymosin-resistant and one plasmin-resistant bovine $\beta$-casein genomic constructs were generated and microinjected to generate transgenic mice. Mouse milk containing each of the three mutant $\beta$-caseins was produced and analyzed by immunoblotting with a bovine $\beta$-casein specific antibody. The mice carrying one of the chymosin-resistant gene constructs produced the milk containing mutant bovine $\beta$-casein with the expected molecular weight. The result of rennin digestion demonstrated that the peptide bond of 192-193 in the mutant protein was chymosin-resistant. However, the mice carrying the plasmin-resistant $\beta$-casein produced two proteins in their milk which reacted with bovine $\beta$-casein antibody and had smaller molecular weights than that of the expected mutant $\beta$-casein.U of I OnlyETDs are only available to UIUC Users without author permissio