期刊
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
卷 33, 期 -, 页码 35-45出版社
WILEY-BLACKWELL
DOI: 10.1042/BA20000069
关键词
antibody; immunoassay; oligosaccharide; recombinant proteins; serology
A truncated form of surface antigen I (SAGIt), the immunodominant surface antigen of Toxoplasmo gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SACI, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG It was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAGIt heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAGIt were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAGI, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG It species or deglycosylation is required in order to use recombinant SAGI as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.
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