UAG Directed Selenocysteine Decoding For The Expression And Characterization Of Glutathione Peroxidase

Oxidative stress,a state of lost balance between the oxidative and anti-oxidative systems of the cells and tissues,results in the over production of oxidative free radicals and reactive oxygen species(ROS).ROS includes hydrogen peroxide,hyphochlorous acid,superoxide anion,singlet oxygen,lipid peroxides,hypochlorite and hydroxyl radical are involved in growth,differentiation,progression and death of the cell.They can react with membrane lipids,nucleic acids,proteins,enzymes and other small molecules.Low concentrations of ROS has an indispensable role in intracellular signalling and defence against pathogens,while,higher amounts of ROS play a role in number of human diseases,including arthritis,cancer,diabetes,atherosclerosis,ischemia,failures in immunity and endocrine functions.GPx is one of the antioxidants that presumably act as safeguard against the accumulation of ROS and their elimination from the system.The active center of selenium-containing glutathione peroxidase(GPx)is selenocysteine(Sec),which is biosynthesized on its tRNA in organisms.The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence(SECIS)to suppress the UGA codon.The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology.In our previously studies,we have succeeded in expression of seleno-containing GPx mutants with all its Cys substituted with Ser in cysteine auxotrophic E.coli strain BL21(DE3)cys-.However,this method is unsuitable for further research of selenoprotein.There are currently several non-SECIS-dependent methods for site-directed incorporation of Sec.A chimeric tRNAUTu compatible with SerRS,SelA and EF-Tu was constructed that enabled Sec-tRNAUTu to be synthesized in vivo and delivered to the ribosomal A-site.tRNAUTu was constructed based on tRNASer by replacing the acceptor stem of tRNASer with that of tRNASer,and the anticodon was changed to CUA to encode UAG as Sec.The adaptation of a chimeric tRNA for the site-specific incorporation of Sec into protein is limited by its binding affinity to SerRS,SelA and EF-Tu.Poor binding of SelA and EF-Tu results in incomplete conversion of Ser-tRNA to Sec-tRNA and the misincorporation of Ser in response to cognate codon.The amber-less E.coli C321.△A.exp has all its endogenous 321 UAG amber codons replaced by UAA and its UAG-specific release factor 1 deleted,thus facilitating amber codon suppression.In this study,we used tRNAUTu in amber-less E.coli C321.△A.exp for the efficient expression of hGPx.Directional optimization of tRNAUTu was performed for optimal Sec decoding at UAG codon and improved the activity of recombined hGPx.The evolved tRNAUTuT6 was used for the expression of GPx3 and its mutants.The non-reducing SDS-PAGE results showed that there are complicated intra-molecular disulfide bonds formed in hGPx3.We found the hGPx3 mutant with all of the Cys mutated into Ser had the highest activity.The detail results are as follows:(1)Efficient expression of hGPx4 with tRNAUTu in amber-less Escherichia coliThe codon UGA for Sec in hGPx4 was mutated into UAG for the incorporation of Sec using tRNAUTu with CUA anti-codon in amber-less E.coli.The hGPx4 expressed has a GPx activity of 18.3 U/mg protein and catalysis following an unsaturation ping-pong mechanism as that of natural hGPx4.MALDI-TOF-MS and ESI-MS analysis results showed that the N-terminal Met was cutted by methionine aminopeptidase after translation.ESI-MS and ESI-MS/MS showed that the hGPx4 expressed contain GPx446Ser,GPx446Sec and Gpx446Glu/Lys/Gln.The intensity of spectrum for each component represented their content and the ratio of Sec was calculated to be 20%to 30%.The yield of hGPx4 was much higher without sodium selenite supplemental than with sodium selenite supplemental,indicated that Ser-tRNAUTu was optimal than Sec-tRNAUTu in suppressing UAG amber codon.(2)Efficient expression of hGPx4 and hGPx1 with improved tRNAUTuT6 in amber-lessEscherichia coliIn order to reduce the mis-incorporation of Ser and natural amino acids at UAG codon,we improved tRNAUTu by substituting the A stem,T stem and T loop with that from tRNASec.Seven tRNAUTy mutants were constructed and all of them could mediate reactive expression of reporter gene FDH and developed purple color.In the further expression and purification of hGPx4 using each tRNAUtu mutant,tRNAUTuT3 failed to express hGPx4.We further expressed hGPx4 and hGPxl using each tRNA mutants and the GPx activities were used to evaluate the efficiency of Sec decoding at UAG amber codon.The results showed that tRNAUTuT1,tRNAUTuT2,tRNAUTuT4 and tRNAUTuT6 were more efficient than tRNAUTu and tRNAUTuT6 could produced hGPx4 with 38.1 U/mg activity and hGPxl with 331.4 U/mg activity,which were both higher than that produced by the other tRNA mutants.The ESI-MS detection of hGPx4 produced by tRNAUTuT6 showed that the spectrum intensity for GPx446Sec was higher than the other component.The ratio of Sec incorporation at UAG amber codon was calculated to be 50%.The steady kinetics study of hGPxl produced by tRNAUTuT6 indicated that it followed a typical ping-pong mechanism similar to natural hGPx1 and reacted with positive cooperativity.The sequence for each tRNAUTu mutants showed the entire antideterminant of tRNASec could be recognized by EF-Tu in tRNAuUTuT2 while it could not be recognizedby EF-Tu in tRNAUTuT3 with a single base different from tRNAUTuT2.Another chimeric tRNAUTuT6,which has a single base change(A59C)compared to tRNAUTu,could produce hGPx1 with GPx activity as high as that produced by tRNAUTuX.These results showed that the base 59 was important for tRNA participating in the synthesis and decoding of Sec.The tertiary structure of tRNASec from different species had a same feather,the formation of tertiary base pair between base 59 in T loop and base 16 in D loop.The base pair is important for the maintaince of D stem and T stem,which interact directly with SerRS,SelA and EF-Tu.(3)Expression and characterization of hGPx3 and its mutants using tRNAUTuT6Plasma glutathione peroxidase(GPx3)belongs to the GPx superfamily and is the only known secreted selenocysteine-containing GPx in human.It exists as a glycosylated homotetramer and catalyzes the reduction of hydrogen peroxide and lipid peroxides depending on the selenocysteine in its active center.In this study,the mature form of hGPx3 without the signal peptide was expressed in amber-less E.coli C321.△A.exp.Reactive hGPx3 able to reduce H2O2 and tBuOOH was produced with high purity and yield.It also could reduce tBuOOH slowly with a low concentration of GSH(10 μM)as that in human plasma.Kinetic steady study of hGPx3 showed that it obeys saturation ping-pong kinetics with respect to GSH,which is different from that of GPx1.Further study showed that the GPx3 exhibited pH and temperature optima of 10 and 50 ℃,respectively.Study on the quaternary structure suggested that the hGPx3 we expressed contained intra-molecular disulfide bridge.Mutational and structural analysis of mutants with three Cys residues individually or jointly replaced with Ser indicates that the formation of intra-molecular disulfide bridge involved structure conformational changes.The structure containing Cys81 and Cys136 were flexible and could form Se-S bridge in relative mutants.This research provides an important foundation for the further catalysis and function study of hGPx3.Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.The involvement of tRNAUTu would provide a new sight into tRNA evolution in uAA incorporation.