Expression, Purification And Characterization Of Peroxiredoxin1and2from Homo Sapiens

Human Peroxiredoxin1（Prx1）and Peroxiredoxin2（Prx2），regarded as importantperoxidase in human body， have been shown to play a key role in protecting cells againstoxidative stress by scavenging reactive oxygen species（ROS）as well as participating in cellsignal transduction. Proteomics research confirmed that Prx1over-expressed in lymphoma，sarcomas and breast cancer cells and Prx2expression were up-regulated in Alzheimer’sdisease，vascular tumors and cervical cancer，which showed a great significance in thediagnosis and therapy of these diseases.Here， human Prx1and Prx2gene were successfully cloned into E.coli BL21（DE3） forrecombinant expression. SDS-PAGE analysis suggested both Prx1and Prx2recombinantprotein were expressed mainly in a soluble form. The recombinant protein were purified byone-step Ni-NTA chelating affinity chromatography with a purity up to88.5%and91.5%，andthe yields were7.59mg/g bacteria and8.82mg/g bacteria，respectively. The Km value forPrx1and Prx2catalysis of the reduction of H2O2are about2.06×10-4M and1.56×10-4M at37°C and pH7.4，as determined by a ferrithiocyanate assay. The ability of Prx1and Prx2toprotect plasmid DNA were tested by utilizing a mixed-function oxidation （MFO） system，and the results showed that Prx1and Prx2can prevent DNA from undergoing oxidative stress.The morphology observation，UV-induced cell apoptosis and ROS assay demonstrated thatPrx2was able to protect NIH/3T3cells from UV-induced damage，however， the protectiveability of Prx1was not obvious. Our antioxidative characterizations of Prx1and Prx2willprovide the basis for their possible applications in cosmetics and other areas.