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.
The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.