Expression Of Bifunctional Enzyme With H GPx2 And ApSOD Activities In UAG Engineered Bacteria

Excessive levels of reactive oxygen species（ROS）are harmful to the body,and the imbalance between the formation and elimination of ROS plays a role in the pathogenesis of many human diseases,such as ischemia/reperfusion injury,atherosclerosis,neurodegenerative diseases,cancer and allergies.Glutathione peroxidase（GPx）and superoxide dismutase（SOD）are important antioxidant enzymes for preventing ROS-induced damage.SOD catalyzes the disproportionation of superoxide anion(O₂^·-)to oxygen（O₂）and hydrogen peroxide（H₂O₂）,and then decomposes H₂O₂ into water by GPx.In addition to their synergistic catalytic functions,these two antioxidant enzymes protect each other to achieve more efficient removal of reactive oxygen species,protect cells from damage,and maintain normal metabolism of reactive oxygen species.Alvinella pompejana source SOD（ApSOD）is a metalloenzyme isolated and purified from alvinella pompejana in seabed hot springs.Its metal cofactors are Cu and Zn.Compared with human SOD（HsSOD）,ApSOD has higher stability and is more suitable for scientific research and medical applications.Human gastrointestinal tract GPx（hGPx2）is a selenoprotein.The catalytic function of selenoprotein is dependent on its active center of selenocysteine（Sec）.Natural Sec is encoded by UGA as a stop codon and is required.A complex coding mechanism can translate UGA inefficiently into Sec.Therefore,it is difficult to obtain selenium-containing GPx by exogenous expression of the cell’s own mechanism.In our previous study,our group used E.coli BL21（DE3）cys auxotrophic system and single protein production（SPP）system to express GPx,and successfully inserted Sec instead of Cys into the peptide chain to obtain higher viability.Selenium GPx,however,this method can only obtain mutants without Cys,coupled with high cost and time consuming,is not conducive to extensive research on selenoproteins.In order to prepare a bifunctional enzyme with synergistic hGPx2 and ApSOD activities,a 21-amino acid flexible peptide Ser（Gly4Ser）*4 was selected as a Linker,in which glycine has the smallest molecular weight and the shortest peptide chain,which can improve Linker’s flexibility,while serine is the most hydrophilic amino acid,which helps the protein remain soluble.The Linker used in this study added 5amino acid GGGGS to the previous study group.The results showed that the extended Linker might better maintain the original structure of the bifunctional enzyme.The hGPx2 and ApSOD genes were first obtained by genetic engineering,and then the two were constructed on the secreted prokaryotic expression vector pRSF Duet-1.Then,the successfully constructed plasmid was co-transformed into UAG engineered bacteria with a plasmid loaded with Sec-incorporated components.Expression was induced in the presence of sodium selenite.UAG engineered bacteria refers to the Amber-less E.coli:C321.ΔA.exp,which removes the release factor 1（RF1）responsible for UAG termination in E.coli and replaces the 321 UAG stop codons in the genome with the UAA stop codon.The engineered bacteria allow UAG to be truly assigned the ability to encode amino acids in this strain.Finally,a bifunctional fusion protein with synergistic hGPx2 and ApSOD activities was isolated and purified by nickel affinity chromatography.The fusion protein has higher GPx and SOD activity.Each enzyme has a specific irreplaceable function that acts in a synergistic manner to better protect the body from damage.Therefore,the design and generation of multifunctional antioxidants will become an important issue in the development of artificial enzymes.We anticipate that synergistic peptidases are expected to be useful in the treatment of human diseases and have potential applications as effective antioxidants in medicine.