Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of beta -lactoglobulin (BLG) were produced in the met hylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of gly codelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28- 30 (S3) of BLG, These three sites were engineered into BLG to produce the v ariants S2, S12 and S123, which carried one, two and three glycosylation si tes, respectively. The oligosaccharides on these BLG variants ranged from ( mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha -linked high mannose type, The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Di gestion of S123 with alpha -1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2), These results indicated a glyc osylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 man nose residues attached mainly by alpha -1,2 linkages. The results also indi cated extension of the Man(5)GN(2) structure by a single alpha -1,6-linked mannose, The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of short er outer chains to the core and no alpha -1,3 outer extensions.