Title

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

Publication Date

4-2001

Abstract

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.

Disciplines

Dairy Science

 

URL: http://digitalcommons.calpoly.edu/dsci_fac/41