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.
Jeng
S Y
SY
Department of Food Science and Human Nutrition, University of Illinois, Urbana 61801, USA.
Bleck
G T
GT
Wheeler
M B
MB
Jiménez-Flores
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002364:Caseins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007768:Lactalbumin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis
D002364:Caseins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007768:Lactalbumin; D002118:Calcium