Prokaryotic Expression And Characterization Of Recombinant Bifunctional Enzymes With Glutathione Peroxidase And Superoxide Dismutase Activities

Reactive oxygen species(ROS)is a general term that broadly describes free and non-free radicals from which oxygen is derived.Oxygen derived free radicals include hydroxyl radicals(·OH),superoxide anions(O2·-)and so on,as well as non-free radicals such as hydrogen peroxide(H2O2),hypochlorous acid(HOCl),and ozone(O3).ROS play a vital role in the growth,development,proliferation and differentiation of multicellular organisms.They are kept in cells at a baseline level that supports cellular proliferation and metabolism,but are also used as key signal transduction molecules that regulate many important metabolic and regulatory pathways in cells.Under normal conditions,there is a complex dynamic balance between the production and elimination of ROS.However,when the balance is broken and excess ROS cannot be removed timely,leading to the reversible oxidation of DNA,proteins and lipids and finally rise up to gene mutation,cancer and a series of other diseases.Organism eliminates different types of ROS by using non-enzymatic antioxidants such as glutathione(GSH),flavonoids,vitamin A/C/E,bilirubin,as well as antioxidant enzymes such as superoxide dismutase(SOD),catalase(CAT),glutathione reductase(GR),glutathione peroxidase(GPx),thioredoxin(Trx),peroxide reductase(Prx)and so on.GPx and SOD can synergize with each other to remove a variety of ROS and protect the body from oxidative damage,meanwhile they can also protect each other.Based on the importance of the synergistic effect,the synthetization of bifunctional antioxidant enzymes with both GPx and SOD activities has higher application value than single-functional antioxidant enzymes.Meanwhile,due to the poor stability and limited sources,the application of natural enzymes is greatly confined.Therefore,how to efficiently express the bifunctional fusion proteinswith high activity and stability is the goal we are pursuing.As a kind of important antioxidant enzyme,eight members of GPx family have been discovered by now.GPx1-4 and human GPx6 belong to seleno-containing GPxs,of which the active center is selenocysteine(Sec).The special encoding mechanism of Sec makes it difficult to express selenoproteins by using traditional genetic engineering methods.The BL21(DE3)cys expression system is a simple and efficient method by using cysteine auxotrophic Escherichia coli to express selenoprotein.The E.coli strain cannot synthesize Cys by itself,it can make use of Sec when Cys was omitted from the culture to express interest protein.It was found in previous studies that the mutation of non-catalytic Cys into Ser can reduce the influence of Sec non-specific substitution.Single protein expression system(SPP system)is a highly efficient protein expression system developed in recent years,in which the ACA sequence of single-stranded RNA can be recognized and cut by using the m RNA interference enzyme Maz F.At this point,the translation machinery of the host cell remains functional,and only interest protein of which the m RNA sequence was recoded to replace the ACA fragment can be produced.A combination of E.coli BL21(DE3)cys auxotrophic strain and SPP system is able to obtain high level and purity interest protein.The UTu expression system is a novel selenium-protein expression system developed in 2012.The Sec-specific insertion can be achieved by synthetic t RNAUTu without the involvement of Sel B and SECIS.In the previous study,our team further optimized t RNAUTu to obtain a new t RNA(t RNAUTu T6),and used the t RNA and amber codon suppression auxotrophic engineering bacteria to express various high activity GPxs successfully.In this study,we successfully obtained a variety of bifunctional fusion proteins containing both GPx and SOD activities through the cysteine auxotrophic expression system and optimized UTu expression system,and the enzymatic properties of fusion proteins were characterized.Furthermore,computer simulations of bifunctional enzyme structure were used to discuss the relationships between structure and function.Besides,the synergy of fusion proteins was tested in vitro experiments.(1)A series of bifunctional fusion proteins were designed based on human GPx1mutant(h GPx1Ser)and alvinella pompejana SOD(Ap SOD)genes.The sequences of h GPx1 Ser and Ap SOD were codon-optimized for high-level expression in the SPP system.All the fusion proteins prepared by using the E.coli BL21(DE3)cys auxotrophic strain and SPP system contain GPx and SOD activities.The enzyme activity of Se-h GPx1Ser-Ap SOD is similar to that of Se-Ap SOD-h GPx1 Ser,indicating that the sequence of GPx and Ap SOD in the fusion protein has little influence on the enzyme activity.During the formation of these fusion proteins,the spatial structures of Ap SOD and GPx may have changed.Therefore,the SOD and GPx activities of the seleno fusion proteins are reduced compared with those of Ap SOD and Se-h GPx1 Ser,respectively.Therefore,we introduced a flexible peptide into the two domains of fusion protein to increase the distance.After the flexible polypeptide was added between h GPx1 Ser and Ap SOD,the activities of both GPx and SOD were enhanced compared with those of Se-h GPx1Ser-Ap SOD.Se-h GPx1Ser-L-Ap SOD showed a high GPx activity of 52.1 U/mg(2130.9 U/?mol),which was markedly improved compared with Se-h GPx1Ser-Ap SOD,and it even could be comparable to those of natural GPx,such as human plasma GPx(302 U/?mol)and bovine liver GPx(2084U/?mol).Meanwhile,the SOD activity of Se-h GPx1Ser-L-Ap SOD was found to be more efficient than most of previous SOD mimics.Kinetic analysis of Se-h GPx1Ser-L-Ap SOD showed a typical ping-pong mechanism,which was similar to those of natural GPxs.The synergism of Se-h GPx1Ser-L-Ap SOD was evaluated by using an in vitro model.(2)Although Se-h GPx1Ser-L-Ap SOD has relatively high activity,there is still a certain gap between it and h GPx1 Ser or Ap SOD.Therefore,by increasing the length of the flexible peptide between two domains,we obtained a new seleno-containing fusion protein Se-h GPx1Ser-l L-Ap SOD.The enzyme activity analysis indicated that its GPx and SOD activity increased by nearly 4 times compared with that of Se-h GPx1Ser-L-Ap SOD.The change of protein quaternary structure is an important reason,as the extension of the flexible peptide enables the fusion protein to exist in the solution as a tetramer rather than a monomer.The Cys resides of Ap SOD domainwere mutated into Ser by site-directed mutation,as expect to improve the SOD activity of fusion protein.To our surprise,the mutation did not alleviate but aggravate the negative impact of non-specific substitution by Sec.The possible reason was that the diselenide bond formed between Sec residues can replace the original disulfide bond and stabilize the protein structure to some extent,while the replacement of Cys by Ser makes the compensation effect invalid.Se-h GPx1UAG-l L-Ap SOD was obtained by using the optimized UTu expression system,and the enzymatic activity was significantly increased compared with Se-h GPx1Ser-l L-Ap SOD.This increase could be explained by structural analysis of fusion proteins based on homology modeling and binding site analysis.The result lead to the hypothesis that the structure of Se-h GPx1UAG-l L-Ap SOD may be optimized by using modified UTu expression system and consequently increase the catalytic efficiency.(3)GPx2,as an important antioxidant enzyme in human body,plays a crucial role in cell proliferation and signal transduction.Multiple bifunctional fusion proteins based on h GPx2 and Ap SOD were constructed by gene recombination technology.And the expression of fusion proteins was conducted by using the cysteine auxotrophic expression system and the optimized UTu expression system.Different from Se-h GPx2 Ser and Se-h GPx2 UAG,all of the fusion proteins produced in this study exist as tetramers.Compared with h GPx1,the activity of h GPx2 is more susceptible to the non-specific substitution of Sec,and the use of optimized UTu system can observably improve the activity of h GPx2 fusion protein.The results obtained from the kinetic analysis demonstrated that it followed a typical ping-pong mechanism similar to that of native GPx.And the synergism of Se-h GPx2UAG-l L-Ap SOD was evaluated by using an in vitro model.Using different subtypes of GPx to construct the bifunctional fusion protein is helpful for us to investigate the feasibility and rationality of the design and construction of bifunctional enzymes comprehensively,and also provides a new direction for the research of multifunctional antioxidant enzymes.Meanwhile,as there is little report on purified native human GPx2,this research is also important for us to improve the understanding of native GPx2.