The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation.
A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.
Biemans
R
R
Service de Génétique Appliquée, Université Libre de Bruxelles, Nivelles, Belgium. rbiemans@sga.ulb.ac.be
Grégoire
D
D
Haumont
M
M
Bosseloir
A
A
Garcia
L
L
Jacquet
A
A
Dubeaux
C
C
Bollen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010843:Pichia / Q000737:chemistry / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000494:pharmacology; D011817:Rabbits; D015996:Survival Rate; D018417:Th1 Cells / Q000276:immunology; D014122:Toxoplasma / Q000276:immunology*; D014123:Toxoplasmosis / Q000175:diagnosis / Q000188:drug therapy / Q000276:immunology; D014124:Toxoplasmosis, Animal / Q000276:immunology / Q000401:mortality / Q000469:parasitology; D014170:Transformation, Genetic
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma