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Protein Engineering, Vol. 14, No. 3, 201-207, March 2001
© 2001 Oxford University Press

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

Chitkala Kalidas1, Lokesh Joshi2 and Carl Batt3,4

1 Field of Microbiology, Cornell University, Ithaca, NY 14853, USA 2 Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA 3 Department of Food Science, Cornell University, Ithaca, NY 14853, USA

Glycosylated variants of ß-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, 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 variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)9(N-acetylglucosamine)2 (Man9GN2) to Man15GN2 and were of the {alpha}-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man9–14GN2. Digestion of S123 with {alpha}-1,2 linkage specific mannosidase resulted in a single product corresponding to Man6GN2. These results indicated a glycosylation pattern consisting of a Man5GN2 structure extended by 4–9 mannose residues attached mainly by {alpha}-1,2 linkages. The results also indicated extension of the Man5GN2 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 shorter outer chains to the core and no {alpha}-1,3 outer extensions.


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