Expression And Purification Of Fusion Protein CTP-SOD In Pichia Pastoris And Antioxidant Capacity Analysis

Superoxide dismutase, which catalyzes the dismutation of superoxide radical into hydrogen peroxide and molecular oxygen, are found in all free-living organisms except some oxygen-sensitive obligate anaerobes. As superoxide radical is involved in the pathogenesis of a variety of diseases, more and more attention have been paid to the clinical application of SOD. SOD should be delivered intracellularly to exert its therapeutic action inside the cytoplasm; however, there is no specific receptor or channel for SOD cross the cell membrane, which greatly reduced its bioavailability.Cytoplasmic transduction peptide (CTP) was a newly designed transduction peptide based on the basic domain of HIV Tat peptide. It could carry the molecules across the cell membrane with a preference to localize in the cytoplasmic compartment. In this study, we used total RNA of human beings to amplify the CTP-SOD fusion gene by reversed transcript PCR. The recombinant plasmid CTP-SOD/pPIC9K was constructed by inserting CTP-SOD fusion gene fragment into Pichia pastoris expression vector pPIC9K which contained AOX1 promoter and secreting peptide, and then transfered into Pichia pastoris GS115 by electroporation. The recombinant GS115/CTP-SOD Pichia pastoris were screened by yeast colony PCR and SDS-PAGE, and then was fermented in flasks. Crude extract was obtained from fermentation centrifugal supernatant by salting out and dialysis. The fusion-protein was purified on a Ni affinity column to more than 90% purity and the pecific activity was 676.5U/mg. The purified protein was stored at-70℃in glycerol buffer until use. We confirmed that the recombinant fusion protein was correctly expressed by using Western blotting assay.To determine whether CTP-SOD can play its biological role in the cells, we have tested the effect of CTP-SOD on cell-viability under oxidative stress. After the HeLa cells were exposed to 60μmol/L pyrogallol without CTP-SOD, only 42.2% of them were viable; the viability increased significantly when CTP-SOD was pretreated in a dose-dependent manner. The cell-viability of HeLa cells pretreated with 1～4μmol/L CTP-SOD increased by approximately 10-30% as compared with that of the control. CTP-SOD significantly improved the survival rate of HeLa cells under the pyrogallol-induced oxidative stress. These results demonstrated that CTP-SOD could cross the cell membrane more efficiently and protect cells from oxidative stress.