Cation-Exchange Purification of Mutagenized Bovine ß-Casein Expressed in Transgenic Mouse Milk: Its Putative Asn₆₈-Linked Glycan Is Heterogeneous

¹ Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801
² Department of Animal Science, University of Illinois, Urbana, Illinois 61801
³ Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, California 93407

Bovine ß-casein (A² genetic variant) was mutagenized to (L70S/P71S) and expressed in transgenic mouse milk. This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X-S/T) (3). Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it. This novel protein was purified from transgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatography (FPLC). The novel ß-casein was separated without cross contamination from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2-ß-mercaptoethanol. The purified (L70S/P71S) ß-casein showed an N-linked oligosaccharide attached to Asn₆₈ and different lectin binding profiles compared with the same protein expressed in yeast. The mouse-expressed ß-casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated ß-casein expressed in Pichia pastoris (4). In addition, the five serine residues located at amino terminus of wild type bovine ß-casein were shown to be normally phosphorylated as in native bovine ß-casein.

Key Words: glycosylated bovine ßbeta;-casein • Mono S-FPLC • transgenic

Submitted on June 21, 1999
Accepted on November 18, 1999