Study On Eukaryotic Expression And Activity Of Antioxidant Enzyme With GPx2 And ApSOD Activities

Reactive oxygen species(ROS)are intermediate products of metabolism and they can be beneficial or harmful to living systems at different physiological concentration.Under the normal physiology condition,its generation and elimination are kept in a balanced state so that it can maintain the relative balance between oxidation and antioxidation.However,ROS can be overexpressed due to various factors such as ecological degradation,environmental pollution and the increasing irregular life of humankind.If the excessive ROS could not be eliminated in time,relevant diseases will become easy-happening and in higher incidence,which will do great harm to human healthy.The process of organism combating oxidation is both complicated and variable.To maintain the balance of physiology system,the synergistic effect of various antioxidases is necessary.By means of synergistic effect and multiple reactions,the enzymatic defense system composed of superoxide dismutase(SOD),catalase(CAT)as well as glutathione peroxidase(GPx)can eliminate redundant chemicals resided in organisms like superoxide anion and Hydrogen peroxide.On one hand,the antioxidant system can provide electron to reduct ROS.One the other hand,it can maintain the balance of ROS in organism by enhancing the antioxidant enzyme activity to accelerate the elimination of ROD.SOD can convert superoxide anion(O2·-)to hydrogen peroxide(H2O2),and then H2O2 is disassembled into H2 O by CAT and GPx.As a result,harmful chemicals like O2·-and H2O2 can be converted into harmless water molecules.Many diseases will occur if the synergistic effect of two antioxidases is disrupted and the damage effect of ROS is amplified.In terms of the interaction of the three chemicals,researchers consider antioxidant enzymes possessing multifunctional activity to be more valuable than single antioxidant enzyme in research and utilization.GPx plays a significant role in the whole antioxidant system due to its highly efficient catalytic ability and its having diverse substrates.The active center of GPx is made up of selenocysteine(Sec)coded by the termination codon UGA.Elaborated and special translation mechanism will be needed for Sec being inserted into protein.As an important antioxidase in organism,SOD is the first lines of defense against free radicals that can O2·-and generate disproportionation reaction,producing O2 and H2O2.Therefore,antioxidant enzyme which possesses both the activity of GPx and SOD has a good research value and broad application prospects.In this work,we obtained recombined fusion protein using GPx2 and ApSOD in eukaryotic expression system.The activity assays confirmed the GPx activity and SOD activity of the the bifunctional enzyme.(1)Design of bifunctional enzymeTo get bifunctional enzyme with hGPx2 and ApSOD activity,hGPx2 gene and ApSOD gene were connected together with gene sequence encoding a pentapeptide(GGGGSGGGGSGGGGS).Gene sequence encoding a His-tag peptide was added to the 3'-terminal of ApSOD for the purification and identification of recombined fusion protein.(2)Construction of p Sel Express1-hGPx2-linker-ApSOD expression vectorGene encoding hGPx2-linker and linker-ApSOD were amplified using PCR method with c DNA library from Hep G2 and p Cold-ApSOD as templates respectively.The entire gene sequence encoding hGPx2-linker-ApSOD was obtained using overlap-PCR method with hGPx2-linker and linker-ApSOD as templates.hGPx2-linker-ApSOD and p Sel Express1 were both digested using Sal I and Xbal I and then ligated together to obtain p Sel Express1-hGPx2-linker-ApSOD.DNA sequencing result verified the construction.(3)Expression and purification of proteinThere are a DNA sequence encoding SECIS downstream of the MCS in p Sel Express1 and directed the encoding of UGA in ORF(open reading frame)as Sec.The plasmid p Sel Express1-hGPx2-linker-ApSOD was transfected into HEK 293 T cells for expression.The recombined protein hGPx2-linker-ApSOD was purified using immobilized metal-affinity chromatography purification system with Ni–NTA resin.SDS-PAGE and Western blot identified the successful of hGPx2-linker-ApSOD expression.(4)Assay of GPx and SOD activityThe activity assays show that the GPx activity of hGPx2-linker-ApSOD was 14.9 U/?mol,15 fold higher compared withGPx mimics ebselen.The SOD activity of hGPx2-linker-ApSOD was 149.3 U/mg.The activity of this bifunctional enzyme was in the same order of magnitude as that of ApSOD expressed in E.coli,but about one order of magnitude lower than that of SOD3.In brief,a bifunctional enzyme with bothGPx and SOD activity was expressed using eukaryotic expression system.This work provided important molecular basis for the research in the synergy of antioxidant enzymes as well as potential medicinal value.