Expression and purification of glycosylated bovine beta-casein (L70S/P71S)in Pichia pastoris

Post-translational glycosylation of bovine beta -casein (L70S/P71S) that re sults in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta -casein expressed in the methylotrophic yeast Pichia pastoris. A mong the growth/induction media used, buffered minimal glycerol (BMG)/buffe red minimal methanol (BMM) media were best for the production of glycosylat ed bovine beta -casein, indicating pH-dependent glycosylation. Glycosylated bovine beta -casein (L70S/P71S) expressed in P. pastoris was purified to h omogeneity by the combination of ammonium sulfate fractionation, Concanaval in A-Sepharose affinity column, and Mono Q anion-exchange FPLC. The purifie d glycosylated bovine beta -casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta -casein was of high-man nose type. Unlike the hyperglycosylation that occurred in yeast, the majori ty of bovine beta -casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta -c asein was normally phosphorylated to the same degree as native bovine beta -casein.