Study On Purification And Water Solubility Of The Uricase From Meyerozyma Guilliermondii

Uricase is an effective drug for the treatment of refractory gout and an important reagent for the clinical determination of uric acid to diagnose hyperuricemia.However,the most suitable pH of the existing commercial uric enzymes is alkaline.The optimal pH of Meyerozyma guilliermondii uricase(MGU)is close to neutral,giving it a unique advantage.1.The recombinantly expressed MGU(R-MGU)was purified using the gel resin Toyopearl SP-650 C and the new material MSP-ZEWB.MSP-ZEWB is a novel hydrophilic magnetic ion exchange material with zwitterionic coat on the surface,which can be charged with different types of net ions on the surface by changing the pH of the medium.Under different sampling pH,R-MGU abundance,saturation of loading R-MGU,the purification multiples,elution efficiency,yields of active fraction and elution volume per unit active of the two materials,under two purification modes of conventional salt-ion competitive displacement elution and ion electrostatic repulsion elution were compared.It was found that MSP-ZEWB was advantageous to Toyopearl SP-650 C in both saturated and unsaturated loading under two elution modes for protein abundances at both ~0.7% and ~1.0%,and which were more pronounced at pH7.6 than at pH8.0.The purification efficiency of MSP-ZEWB was insignificantly changed after repeated regeneration for 16 times.The optimum pH was 7.2 for the enzyme purified by MSP-ZEWB and verified by SDS-PAGE.2.In order to guide the discovery of mutants with higher water solubility,a method of rapid characterization of protein solubility by resonance light scattering(RLS)was established.The RLS signal response curve was obtained by synchronous scanning mode,and the maximum concentration of each protein dispersion state was predicted according to the intersection point of the response curve,and the solubility of this state was defined accordingly.By comparing the solubility determinations of bovine serum albumin(BSA),under different pH and salt ion,and the solubilities of glutathione S-transferase isoenzymes alpha,M?(GSTA,GSTM),RLS was proved to estimate the solubility of protein.There were two intersection points in the RLS response curves of the tested proteins,among which the lower one can be the approximation of the maximal concentraion or the solubility of the protein in the single molecular dispersion,and the higher one was the protein aggregation of multiple molecules.Both intersection points of BSA(isoelectric point 4.6)increased with the increase of pH(6.5-7.4),and the high concentration intersection points were close to the solubility of commodities under the same conditions.The intersection point concentration decreased with the increase of NaCl concentrations.The concentration intersection points of RLS response curve of GSTA and GSTM were smaller than BSA,supporting the method has universality.The two intersection points of the R-MGU response curve corresponded to 0.24g/L and 0.30g/L respectively,much lower than those tested proteins.RLS can directly characterize the solubility of protein macromolecules.This method is simple,sensitive and needs less comsuption of proteins,which has an unique advantage for rapid comparison of solubility of proteins/mutants in low abundances.3.In order to design mutants with improved solubility,homology modeling was used to select the homology template with the highest sequence similarity for MGU(3GKO.pdb).The structure of MGU was superimposed with Bacillus fastidious uricase(BFU,4R99.pdb)of high solublility,looking for the surface hydrogen bond in MGU at the position corresponding to that of the surface ionic bond in BFU crystal.The surface hydrogen bonds were mutated into ionic bonds.After comparing the structures,three mutants of S96 D,T241D and L246 D were screened.After the purity verification by SDS-PAGE,RLS intensity were determined.Compared with R-MGU,the water solubility of S96 D was increased by about 170%,T241 D by about 250%,and L246 D by about 290%.The water solubility of mutants were successfully improved comparing with MGU.4.To find the structural determinants of optimal pH by omparing with V144 A mutant of BFU.BFU has higher specific activity and good thermal stability,but its optimum pH is 9.2.The Superimposition of the three-dimensional structures showed that the arginine at position 272 on BFU(4R99.pdb)and the histidine at position 261 on MGU were close in space.V144 A of BFU was used as a wild-type template.Synergistic mutation of arginine in V144 A at position 272 to histidine and lysine,tyrosine and threonine,glutamate,respectively,for characterization of their optimum pH,whereas no changes in the optimum pH of 9.2 were observed.In summary,we proposed a purification scheme of R-MGU,developed a method for quickly characterizing the solubility of mutants,and successfully obtained R-MGU mutants with improved water solubility.