Study On Prokaryotic Expression And Activity Of Bifuctional Enzyme With GPx4 And ApSOD Activities

In the process of physiological metabolism, a large amount of reactive oxygen species（ROS） are produced. However, variety concentration of reactive oxygen species has harmful as well as beneficial effects on the body. At a low concentration,ROS play an important role in cell signal transduction, stimulating cell proliferation,inducing cell apoptosis and so on. At certain pathological conditions, a large amount of ROS are produced and cannot be timely cleared by the body. It will have serious side effects on the body, such as the destruction of the intracellular protein and DNA.There is an important oxidation and antioxidation physiological balance system in the body containing Glutathione peroxidase（GPx）, superoxide dismutase（SOD） and catalase（CAT）. The function of this system is clearing redundant reactive oxygen species and repairing the biomacromolecules damaged by ROS, keeping the state of ROS at a physiological level. The complicated process of antioxidation needs multiple antixoidases communicate with each other in perfect union. The study of single functional antioxidases cannot satisfy the need people exploring the synergistic effects between antioxidases, so it is imperative to study multi-functional antioxidant enzymes.To prepare synergistic bifunctional enzyme with GPx and SOD activity, GPx4 mut and Ap SOD gene connected together by gene sequence encoding a flexibility linker GGGGS was cloned into p Cold I expression vector and transformed into cysteine-auxotrophic expression system. Finally, recombined protein GPx4_mut-linker-Ap SOD was expressed after IPTG induction. Cys in GPx4 mut has been successfully replaced with Ser by Doctor Yu yang in our group（ Cys2/10/37/66/75/107/148Ser）, using quick-change site-directed mutagenesis.（1） Design of bifunctional enzymeTo get bifunctional enzyme with GPx4 and SOD activity, GPx4 mut gene and Ap SOD gene were connected together with a pentapeptide（GGGGS） gene.（2） Construction of p Cold I-Ap SOD expression vectorp UC-Ap SOD gene synthesized by Shanghai biological engineering company was digested by restriction endonuclease Ecol RI/Xbal, and then ligated into p Cold I digested by the same restriction endonuclease, yielding p Cold I-Ap SOD.（3） Construction of p Cold I-GPx4_mut-linker-Ap SOD expression vectorA DNA sequence encoding flexibility linker GGGGS was added to the 3’terminal of GPx4 mut using a 3’ primer by PCR with p Cold I-GPx4 mut as template.The PCR product GPx4_mut-linker was digested by restriction endonuclease Nde I/Bam HI and ligated into p Cold I-Ap SOD digested by the same restriction endonuclease, yielding p Cold I-GPx4_mut-linker-Ap SOD.（4） Expression and purification of proteinp Cold I-GPx4_mut-linker-Ap SOD expression vector was transformed into cysteine-auxotrophic Escherichia coli expression system BL21（DE3） cys, obtaining expression strains BL21（DE3） cys-p Cold I-GPx4_mut-linker-Ap SOD. After that,plasmid p Mza F was transformed into the expression strains. The cell colony was grown at 37℃ until OD600 reached 1.2 following inducible expression for a night at 15 ℃, cells were collected and lysed, recombined protein was purified by Ni²⁺chelating chromatography.（5）Assay of GPx and SOD activityThe results show that the SOD activity of Se-Cu/Zn-GPx4_mut-linker-Ap SOD was 86.2U/mg, while that of nonseleno-Cu/Zn-GPx4_mut-linker–Ap SOD was135.8U/mg and that of nonseleno-Cu/Zn-Ap SOD was 454.8U/mg. The GPx activity of Se-GPx4_mut-linker-Ap SOD was 6.1 U/mg, lower than that of Se-GPx4_mut（21.5U/mg）, but still exceeding than that of small molecular simulation Ebselen.In summary, a bifunctional enzyme with GPx and SOD activity was constructedby genetic engineering method. The obtain of this bifunctional antioxidases not only has great significance on the illustration of catalytic mechanism and synergistic effect, but also has potential pharmaceutical value.