Functional Study Of Human Glutathione Peroxidase-7via Prokaryotic Expression And Purification

Despite Glutathione peroxidase7(GPx7) has been shown to have littleGPx activity in vitro, loss of the GPx7in cellular level or GPx7knockoutmice has triggered off accumulation of intracellular ROS and oxidativeDNA damage which eventually lead to progression towards carcinoma orsystemic oxidative stress. All these results may imply that GPx7showedGPx activity distinct from in vitro. Consequently, the nature cellularfeature of GPx7prompted us using human GPx7, which expressed throughprokaryotic expression system, for alleviating ROS in extra cellularenvironment and eventually exerting suppression of tumor growth andpromoting effect in myeloid regeneration after chemotherapeutics injuredis a novel aspect and deserving further investigation. Here, this thesisnarrated the construction of GPx7expression vector, expression andpurification of GPx7, effect of GPx7with tumour cell growth rates andbone marrow regeneration in chemotherapeutic injured mouse model andfinally intra and extra cellular activity of GPx7towards ROS (especiallyH2O2).Human GPx7coding domain sequence was cloned into the pET24avector and expression of GPx7in E.coli BL21(DE3) is of soluble form. Apractical GPx7purification process included two chromatography steps inwhich cation exchange (Sulfopropyl media) and size exclusion (Superdexmedia) chromatography was adopted. The purity of resulting pure productwas examined through SDS-PAGE and the result showed that only onespecies was visible in SDS-PAGE. Western blotting identification ofpurified end product also proved that it was GPx7. In vitro expression of GPx7in A549cell lines (lung epithelial tumour cell) also reduced growthrates (p<0.05) of A549cancer cell lines. Besides, subcutaneously injectionof GPx7also showed reduction of tumour weight in xenograft mousemodel (p<0.05) and the inhibition rate was29.26%. Subcutaneouslyinjection of GPx7to mouse model of chemotherapeutic-injured bonemarrow also showed promoting effect on bone marrow regeneration duringthe9th,11thand13thday (p<0.05) and eventually increased the amount ofwhite blood cell and platelet cell. This result indicated that GPx7may exertits ROS scavenging function through alleviating damage of ROS, whichgenerated by the chemotherapeutic agent, to myeloid cell. Meanwhile,ROS level was also decreased significantly in GPx7-expressing A549cell,indicating that ROS was involved in A549cell growth and suppression ofA549cell growth with over expression of GPx7may due to regulation ofROS level by GPx7.