Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris.
Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results 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. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation. Glycosylated bovine beta-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 beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.
Choi
B K
BK
Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801, USA.
Jiménez-Flores
R
R
D000818:Animals; D015153:Blotting, Western; D002364:Caseins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D004587:Electrophoresis, Agar Gel; D005260:Female; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D008970:Molecular Weight; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D002364:Caseins; D011994:Recombinant Proteins