Expression,Purification And Structural Function Of Recombinant Urate Oxidase

Urate oxidase(UOX),also known as uricase,is an oxygen oxidoreductase involved in the purine degradation pathway.It converts urate to allantoin which is more soluble than uric acid and excreted easily by the kidney.And it has been proved that active uricase is absent in humans due to gene mutations.As a consequence,uric acid levels are higher in human than in other mammals possessing a functional uricase,leading to hyperuricemia.Long-term hyperuricemia causes a number of disorders,including gout and urate nephropathy associated with tumor lysis syndrome.Studies have also shown that hyperuricemia in humans can increase the risk of cardiovascular diseases,chronic nephropathy,hypertension and stroke.Therefore,the introduction of exogenous uricase is an ideal way to treat hyperuricemia.In addition to poor stability,short half-life,the current clinical application of uricase is greatly limited since it is a foreign protein with immunogenicity.In order to obtain the uricase with good stability and low immunogenicity,we study the structural characteristics of Candida utilis uricase and modify the amino acid sequence of prototype uricase,then express and purify recombinant uricases through prokaryotic expression system.Otherwise,the relevant structural properties of recombinant proteins are confirmed to lay the foundation for lucubrating.Protein structure is closely related and to a certain extent determines the function of protein,so understanding protein structure is a prerequisite for functional research.In view of the fact that there is no report about the structure of Candida utilis uricase,we use homologous modeling to obtain the three-dimensional structure,and analyze the structural characteristics to modify uricase amino acid sequence.Moreover,with the rapid development of biological structure and bioinformatics,some softwares for predicting T cell epitopes come into being,which are applied to predict and further remove the uricase MHC molecular antigen binding peptide.In this study,the genes of the prototype and modified uricase are synthesized and cloned into pET-22b(+)to construct expression vector pUOX,then transformed into Escherichia coli BL21(DE3).Five recombinant uricases,including the prototype of uricase and four modified uricases,are high-level expressed successfully in a complete soluble form.After Q sepharose FF and gel filtration chromatography purification,the recombinant uricase proteins with high purity are obtained.The enzyme activity assay shows that the recombinant uricases have better specific activity.The purified recombinant uricases are prepared to study structural confirmation and catalytic activity.Mass spectrometry measures the molecular weight and the peptide mass profile of the recombinant uricase,which identifies the recombinant proteins with complete amino acid sequence and primary structure.Coupled multi-angle static light scattering gel chromatography and high performance liquid chromatography experiments confirm that the recombinant uricases are homologous tetramer,and relative to the modified uricase,the prototype uricase easily forms a higher polymer,with poor stability.Further SDS-PAGE experiment shows that the formation of the polymer may be due to the cysteine residues being oxidized into disulfide bond.The catalytic activity of the uricase in different pH conditions and the Michaelis-Menten constant Km are measured by enzyme-substrate reaction,demonstrating that recombinant uricase have high affinity for uric acid.The recombinant uricase CUOX-D1 is detect on physicochemical and biological activity.Non-reducing SDS-PAGE electrophoresis shows the purity is about 98.59%.The maximum absorption peak at 280 nm,and the isoelectric point is 8.15-8.65.N-terminal is identical to the theoretical amino acid sequence.The results of mass spectrometry are consistent with the theoretical amino acid sequence.The specific activity of recombinant uricase is 3.20U/mg,which is similar to that of uricase archetype.In conclusion,we successfully construct the expression system of recombinant uricase.Establishing the purification process initially,and detect on physicochemical and biological activity.It lays the foundation for future modifying to improve the stability and reduce the immunogenicity of uricase.