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

被引:14
作者
Choi, BK
Jiménez-Flores, R [1 ]
机构
[1] Calif Polytech State Univ San Luis Obispo, Dept Dairy Sci, San Luis Obispo, CA 93407 USA
[2] Univ Illinois, Dept Food Sci & Human Nutr, Urbana, IL 61801 USA
关键词
glycosylated bovine beta-casein; Con A column; Mono Q FPLC; Pichia pastoris;
D O I
10.1021/jf001298f
中图分类号
S [农业科学];
学科分类号
09 ;
摘要
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.
引用
收藏
页码:1761 / 1766
页数:6
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