Cation-exchange purification of mutagenized bovine beta-casein expressed in transgenic mouse milk: its putative Asn68-linked glycan is heterogeneous.
Bovine beta-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 beta-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-beta-mercaptoethanol. The purified (L70S/P71S) beta-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 beta-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 beta-casein expressed in Pichia pastoris (4). In addition, the five serine residues located at amino terminus of wild type bovine beta-casein were shown to be normally phosphorylated as in native bovine beta-casein.
Choi
B K
BK
Department of Food Science and Human Nutrition, University of Illinois, Urbana 61801, USA.
Bleck
G T
GT
Jimenez-Flores
R
R
D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D015151:Immunoblotting; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000737:chemistry*; D010766:Phosphorylation; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry*
D002364:Caseins; D011134:Polysaccharides