Expression Of Bifunctional Enzymes Containing GPx2 And ApSOD Activities In Cysteine Auxotrophic Expression Bacteria

Oxidative stress is a state of imbalance between the oxidation and antioxidant systems of cells and tissues,resulting in excessive oxidation of free radicals and reactive oxygen species?ROS?.Excessive production of ROS may attack cellular proteins,lipids and nucleic acids,leading to cell dysfunction,including loss of energy metabolism,changes in cell signaling and cell cycle control,genetic mutations,changes in cellular transport mechanisms,decreased biological activity,immune activation and inflammation.The oxidation-antioxidant balance system in vivo can modulate the oxidative stress state.Endogenous antioxidants mainly include several enzyme antioxidants such as glutathione peroxidase?GPx?,superoxide dismutase?SOD?,and catalase?CAT?.Several antioxidant enzymes in the organism work synergistically to maintain ROS at a stable level.For glutathione peroxidase?GPx?,most of the highly active GPx is selenium-containing enzyme,and the catalytically active center selenocysteine?Sec?is encoded by the stop codon UGA,so a special mechanism is needed to incorporate Sec.In proteins,since the eukaryotic and prokaryotic organisms differ in the insertion mechanism,it is difficult to express selenium-containing GPx using traditional genetic recombination techniques.Since GPx is an important selenium-containing antioxidant enzyme,many researchers have prepared GPx mimics,but there are major problems in activity and preparation methods.This experiment uses the E.coli BL21?DE3?cys auxotrophic system,which is an effective method for the preparation of selenoproteins in recent years.Its basic principle is to use Cys transport RNA?tRNACys?to bind Sec,when there is lack of Cys.So it allows Sec to be successfully incorporated into the newly expressed protein.In order to express recombinant proteins more efficiently,this experiment also used the SPP expression system,which can reduce the cost and ensure the protein expression yield.In combination with the E.coli BL21?DE3?cys auxotrophic system and the SPP system,we are gradually increasing the efficiency of selenoproteins production.In order to study the synergistic effects of various antioxidant enzymes in vivo and increase the activity of antioxidant enzymes,we simultaneously expressed superoxide dismutase?SOD?and prepared a bifunctional enzyme containing GPx activity and SOD activity.To prepare antioxidant enzymes with bifunctional activity of GPx2 and ApSOD,we selected a flexible linker,ligated the GPx2 and ApSOD genes together and cloned into the pColdI expression vector,followed by the E.coli BL21?DE3?cys auxotrophic system.The recombinant GPx2-linker-ApSOD protein was directly prepared.?1?Design of gene sequences encoding bifunctional enzymesWe selected a flexible peptide linker to link the GPx2 gene and the ApSOD gene,and successfully obtained a coding gene capable of fusing GPx2 and ApSOD.?2?Construction of pColdI-GPx2-linker-ApSOD plasmidPrimers were designed by the software,and linker was ligated to GPx2 by PCR to obtain GPx2-linker gene,and then PCR was used to construct linker-ApSOD.Finally,the entire fragment of GPx2-linker-ApSOD was obtained using overlapping PCR.The target gene GPx2-linker-ApSOD and the vector pColdI were digested with the endonuclease NdeI and XbaI,and then use the ligase to construct the pColdI-GPx2-linker-ApSOD gene.The gene was transformed into DH5?bacteria and the plasmid was extracted.?3?Expression of pColdI-GPx2-linker-ApSOD protein and purificationThe pColdI-GPx2-linker-ApSOD plasmid and the pMzaF plasmid were transformed into BL21?DE3?Cys auxotrophic Escherichia coli to obtain pColdI-GPx2-linker-ApSOD in E.coli BL21?DE3?cys auxotrophic system.Sec is added during the culture to make it a selenoprotein.The target protein was purified by Ni²⁺column chelate chromatography,and the heteroprotein was washed off by many kinds of imidazole to finally obtain the target protein.?4?Determination of GPx and SOD activity of pColdI-GPx2-linker-ApSOD proteinThrough multiple viability measurements,the SOD activity of pColdI-GPx2-linker-ApSOD was 109.3±11.0 U/mg,and its GPx activity was 3.7±0.4 U/mg.In summary,this experiment successfully constructed a bifunctional enzyme with GPx2 and ApSOD activity.This genetic engineering method for constructing a bifunctional enzyme not only solves the problem of limited source of natural antioxidant enzymes,but also reduces production cost and increases protein yield.The increase in vitality also lays a good foundation for the research in the future on the synergistic mechanism of various antioxidant enzymes.