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Biotechnology and Applied Biochemistry (2001) 33, (35–45) (Printed in Great Britain)

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Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation

Odile Letourneur¹, Gaspard Gervasi, Sébastien Gaïa, Joëlle Pagès, Bénédicte Watelet and Michel Jolivet

bioMérieux, R & D, Unité Immunoessais, Chemin de l'Orme, 69280 Marcy l'Etoile, France

Abbreviations used: amu, average mass unit; BMGY, rich standard medium containing glycerol; BMMY, rich standard medium containing methanol; DIG, digoxigenin; ELFA, enzyme-linked fluorescent assay; FBS, fermentation basal salts; G418, geneticin; GNA, Galanthus nivalis agglutinin; GPI, glycosylphosphatidylinositol; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; LC-ESI-MS, liquid chromatography coupled with electrospray ionisation MS; SAG1, surface antigen 1; SAG1t, truncated form of SAG1.

¹ To whom correspondence should be addressed (e-mail odile_letourneur@eu.biomerieux.com).

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, 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 SAG1t 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 SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing a1-2-, a1-3- or a1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, 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 SAG1t species or deglycosylation is required in order to use recombinant SAG1t 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.

Received 15 September/24 October; accepted 31 October 2000

Portland Press Ltd © 2001