Optimization for high-level expression in Pichia pastoris and purification of truncated and full length recombinant SAG2 of Toxoplasma gondii for diagnostic use.
SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma gondii. In the present study, truncated recombinant SAG2(S) and full length recombinant SAG2(T) of T. gondii were optimally produced (approximately 15 mg/liter) in Pichia pastoris expression system using BMMY medium at pH 3, 25 degrees C in 0.5-1% methanol and a time-course of 1-2 days. The recombinant proteins were purified using a commercial gel filtration purification system obtaining approximately 33% recovery. The purified SAG2(S) and SAG2(T) showed molecular masses of 45 and 36 kDa by SDS-PAGE, respectively. The recombinant proteins were evaluated by Western blotting with patients' sera and demonstrated 90% sensitivity and 100% specificity for detection of toxoplasmosis. This study provided a means for large-scale expression and purification of SAG2, which should be useful for diagnosis of toxoplasmosis.
Ling
Lau Yee
LY
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. lauyeeling@um.edu.my
Ithoi
Init
I
Yik
Fong Mun
FM
D000953:Antigens, Protozoan / Q000096:biosynthesis*; D015153:Blotting, Western; D003001:Cloning, Molecular; D046508:Culture Techniques; D006801:Humans; D010843:Pichia; D015800:Protozoan Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D012698:Serologic Tests; D014122:Toxoplasma / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis*
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C067547:surface antigen P22, Toxoplasma