Research On Human -Dog Chimeric Uricase Construction、expression、 Purification And Characterization

Objective: To construct Escherichia coli expression system of Human- dog chimeric uricase,choose the high level expression strain andand obtain the recombination protein of uricase.To establish fermentation and purification pilot process of uricase and study its properties.To become drug candidates for treatment of gout.Methods:(1) The high level expression of uricase in Escherichia coli①Use of genetic engineering, firstly, The vector pET-3c was digested by restriction enzyme BamHI and Nde I, Then the GeneSynthesis of uricase inserted into the pET-3c toconstruct the recombinant plasmid pET -3c-Uricase.②recombinant plasmid pET-3c-UHC transformed into expression host strain E.coli BL21 (DE3) Star plysS.③The multiple clones were obtained by LA plates whichcontains increasing concentrations of Amp④flask expression and choose the high level expression strain.(2) Research uricase fermentation:Optimization of fermentation conditions for uricase through different media, different induction temperature, induction dose and time(3)purification of uricase: The protein was purified by saturated ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography .(4) properties of uricase①physical and chemical properties of uricase②To study activity and stability of uricase by different temperature and pH valu③To study uricase enzyme kinetics.Results:(1) uricase have achieved Soluble expression in E. coli, The expression level reached 30%.(2) The best fermentation conditions: medium M9-YE-Glu; induced temperature 30℃; induction time of 3 hours; induction dose of 0.2mM IPTG.(3) The purification process is stable and reproducible.the purity of uricase are More than 95%, the recovery are about 60%. Established Quality control method for purification.(4) SDS-PAGE and mass spectrometry detection of uricase molecular weight was consistent with the theory.. SEC-HPLC analysis indicated that uricase four identical subunits.(5) uricase enzymatic reaction optimum pH of 8.6, optimum temperature was 37℃.