Odile Letourneur

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

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 α1‐2‐, α1‐3‐ or α1‐6‐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.

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellmans reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

Erratum to “Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris”: [J. Virol. Methods 99 (2002) 99–114]

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellmans reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.