Expression and Purification of Glycosylated Bovine β-Casein (L70S/P71S) in Pichia pastoris

 University of Illinois.

 Present address:  Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6049.

 Author to whom correspondence should be addressed [telephone (805) 756-6103; fax (805) 756-2998; e-mail rjimenez@oboe.aix.calpoly.edu].

 California Polytechnic State University.

Post-translational glycosylation of bovine β-casein (L70S/P71S) that results in Asn₆₈-linked glycan on the protein was obtained in up to 30% of total β-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine β-casein, indicating pH-dependent glycosylation. Glycosylated bovine β-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A−Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine β-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated β-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine β-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine β-casein was normally phosphorylated to the same degree as native bovine β-casein.

Keywords: Glycosylated bovine β-casein; Con A column; Mono Q FPLC; Pichia pastoris