Expression And Characterization Of Novel Bifunctional Antioxidant Enzymes And Exploration Of A New Encoding Method For Sec In Vitro

The redox balance is destroyed when the body is exposed to uncontrollable factors,leading to excessive production of highly reactive oxygen species（ROS）,thereby inducing damage to the body.The antioxidant defense system plays a crucial role in eliminating ROS and suppressing their toxic effects.Antioxidant is a complex process that requires the interaction of multiple enzymes.Superoxide dismutase（SOD）can dismutate the superoxide anion(O₂^·-)into hydrogen peroxide（H₂O₂）,which could be reduced to non-toxic water（H₂O）by glutathione peroxidase（GPx）.Therefore,in vitro expression or simulation of a single enzyme cannot provide a thorough understanding of the antioxidant system,fusion proteins with multiple enzyme activities obtained in vitro can synergistically clear multiple ROS and lay a good foundation for the clinical application of antioxidant enzymes.Selenocysteine（Sec）,which is the key to efficiently removing ROS for GPx,is encoded by UGA,which is usually used as a stop codon.Sec has a complicated encoding mechanism,which greatly limits the acquisition of Se-GPx in vitro.The coding of Sec in vitro has long attracted much attention.The UTu expression system realizes the site-specific insertion of Sec into the protein,but its insertion efficiency is not very ideal.In the previous study,a new hybrid tRNA^UTuT6 with higher decoding efficiency by optimization of tRNA^UTu was developed and expressed in E.coli C321.△A.exp to obtain a recombinant GPx with higher activity.Presently,reprogramming of UAG is the main method for site-specific incorporation of Sec into proteins in vitro using hybrid tRNA.Encoding codons other than UAG into Sec can provide more blank codons for the expression of selenoproteins in vitro and contribute to the development of new methods for obtaining selenoproteins in vitro.In recent years,the use of quadruplet codons to encode unnatural amino acids（UAAs）has attracted extensive attention,but so far,it has not been reported that the quadruplet codons can be used to encode Sec.Based on the above research background and basis,in this study,several novel fusion proteins were obtained by using tRNA^UTuT6in E.coli C321.△A.exp.Meanwhile,their synergistic antioxidant capacity has been confirmed in in vitro peroxidation damage models.Next,based on tRNA^UTuT6,the anticodon was mutated into UCUA,UCCU,and CCCU（decoding UAGA,AGGA,and AGGG）,and the mutation was introduced into the anticodon ring,and then,several mutant tRNAs were constructed to realize the in vitro encoding of Sec.The experimental results are as follows:1.Expression and characterization of novel bifunctional antioxidant enzymes withGPx and SOD3 activitiesThe genes of human GPx1（hGPx1）/human GPx4（hGPx4）,and human SOD3（h SOD3）active domain（h SOD3-72P）were connected by different length Linkers,and then several novel bifunctional proteins were obtained using tRNA^UTuT6 in E.coli C321.△A.exp.The quaternary structure of fusion proteins is the same as that of natural GPx.The results showed that there was almost no difference in GPx activity between the hGPx1 fusion proteins(Se-hGPx1_UAG-L₃-SOD3-72P:190±29U/mg,Se-hGPx1_UAG-L₄-SOD3-72P:199±16U/mg).Although there is room for improvement compared to natural proteins,they are still the bifunctional fusion proteins with the highest GPx activity to date.The results of the enzymatic properties showed that the fusion protein of hGPx4 had stronger alkali resistance and high temperature resistance.The structural change of fusion protein and the formation of disulfide bonds by free Cys in hGPx4were one of the reasons for the enhanced resistance of Se-hGPx4_UAG-L₃-SOD3-72P protein to the external environment.The fusion proteins catalyze the reduction of peroxide in ping-pong mechanism,which is the same as the natural GPx.We confirmed that Se-hGPx1_UAG-L₄-SOD3-72P bifunctional protein has synergistic antioxidant capacity by using in vitro peroxidation damage models.2.Exploration of a new encoding method for Sec in vitroIn the previous study,not only recombinant GPx with higher activity,but also bifunctional enzymes with bothGPx and SOD activities were obtained using tRNA^UTuT6 by decoding UAG as Sec.To explore whether codons other than UAG can also encode Sec in vitro,the triplet anticodon of tRNA^UTuT6was mutated to a quadruplet anticodon and proved for the first time that,in addition to UAG,the quadruplet codons can also encode Sec.The effectiveness of the decoding rate can be enhanced through mutations introduced in the tRNA anticodon ring,thereby establishing a reliable foundation for expressing selenoproteins in vitro.In this study,several novel bifunctional antioxidant enzymes with bothGPx and SOD activities,which can play a synergistic role,were obtained in E.coli C321.△A.exp using tRNA^UTuT6,laying a foundation for the development of therapeutic drugs for oxidization-related diseases.In addition to the commonly used UAG codon,the quadruplet codons can also be used for Sec encoding in vitro,which is helpful for the development of a new expression method for selenium protein in vitro.