Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris.
Glycosylated variants of beta-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) (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). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated 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 shorter outer chains to the core and no alpha-1,3 outer extensions.
Kalidas
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Field of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Joshi
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Batt
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D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry*; D014644:Genetic Variation; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics*; D008358:Mannose / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D010843:Pichia / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011257:Pregnancy Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D016415:Sequence Alignment
D005656:Fungal Proteins; D006023:Glycoproteins; D007782:Lactoglobulins; D009844:Oligosaccharides; D011257:Pregnancy Proteins; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; D008358:Mannose