Preparation And Crystallographic Study Of The Peroxiredoxins From Schistosoma Japonicum

Schistosomiasis existed in tropical and sub-tropical countries of Africa, Asia and South America has a greatharmful to the people’s health. This disease which has been become a worldwide public health problem is causedby pathogens of blood. Schistosoma japonicum, a causative agent of schistosomiasis, is a parasitic diseaseaffecting millions of people in China. Current treatment relies on a single drug of Praziquantel, however, therapybased on just a single compound is at risky because of the high probability to cause resistance. Indeed,schistosome strains displaying low praziquantel susceptibility have already been reported. The Study showed thatSjPrxs play an important role in the detoxification of reactive oxygen species (ROS) in S. japonicum and it maybe a potential vaccine candidate or new drug target for schistosomiasis.Antioxidant peroxidase (SjPrxs) in Schistosoma japonicum includes homology of SjPrx1, SjPrx2andSjPrx3. According to the amino acid sequence only SjPrx3contains the mitochondrial targeting sequence. SjPrx1may work as a scavenger against reactive oxygen species (ROS) generated outside of the schistosomes to preventthe oxidation of the bodies and/or the attack by immune cells producing the ROS. SjPrx2plays important roles inintracellular redox signaling and/or in the reduction of ROS generated through the hemoglobinolytic process inthe digestive tract. SjPrxs from schistosoma japonicum, a kind of important antioxidant enzymes in the body, is apotential schistosomiasis vaccine candidates and new drug targets.This study contained the cloning, expression,purification of typical2-Cys peroxidase in S. japonicum (SjPrxs) and the preliminary structural study of SjPrx1.The main research results include:(1) The recombinant vector synSjPrx1-pET28a and wtSjPrx1-pET28a containg N-His tag were transformedin E.coli BL21(DE3). SjPrx1was expressed as a soluble protein with molecular weight of25kDa. The optimizedexpression condition was OD600=1.0, IPTG concentration0.8mM,24℃and18h. The recombinant protein wascaptured by Ni2+and following DEAE affinity chromatography. Experiment showed that synSjPrx1had enzymeactivity. The final concentration of synSjPrx1was up to42mg/mL with purity higher than95%. The synSjPrx1crystals was obtained in the buffer of0.2M NH4Cl,0.2M Tris-HCl, pH7.5,0.025M CaCl2and the diffractionresolution was about3.05. SynSjPrx1structure was solved and three molecules were found in the symmetricunit, however, the electronic map of eighteen residues in C-terminal were invisible and can not be modeled.(2) Recombinant vector of wtSjPrx1-pET42a containing6×His Tag in the C-terminal was constructed andtransformed into E.coli BL21(DE3). Protein isolation, purification and preliminary crystallographic researchwere carried out according to the methods above. We got the single wtSjPrx1crystals under the conditions of1.0M Ammonium sulfate,0.1M HEPES pH7.0,0.5%Polyethylene glycol8000and0.1M Sodium malonate pH7.0,12%Polyethylene glycol3350. X-ray diffraction and structure determination were in process.(3) Recombinant plasmid of wtSjPrx2-pET28a and wtSjPrx3-pET28a were constructed and expressed in E.coli BL21(DE3). The optimized expression conditions of wtSjPrx2and wtSjPrx3are OD600=1.0,0.8mM IPTG, 24℃,200rpm,18h and wtSjPrx3OD600=1.0,0.7mM IPTG,24℃,200rpm,18h, respectively. These two targetproteins were soluble with Mw of26kDa for wtSjPrx2and27kDa for wtSjPrx3. The recombinant proteins werecaptured via two-step and the purity is higher than90%.