Recombinant Construction And Screening Of Mammalian Uricase And Structure And Function Of Cys Residues

Uricase（EC 1.7.3.3）is a key enzyme in purine metabolism,which can change slightly soluble uric acid into soluble allantoin.It has been used in the treatment of hyperuricemia and gout.Because of its high homology with putative human uricase,mammalian uricase has become the focus on the development of uricase drugs.Cysteine（Cys）,which forms disulfide bonds,are key amino acids that influence the higher structure and function of proteins.Mammalian uricase protein contains four Cys residues.However,there are no studies on the structure and function of mammalian uricase free Cys.In this thesis,we used the canine uricase（CU）as a template to study the constitution form of mammalian uricase Cys.The structure of mammalian Cys residues and its effect on protein stability were also studied.Based on DTNB colorimetry,for the first time,we found that all four Cys residues of uricase,represented by CU,were free and did not participate in disulfide bond formation.After Cys residue was blocked,the enzyme activity of CU was completely lost,which indicated that the redox form of Cys residue had great influence on the enzyme activity.Therefore,we successfully constructed the mutant strain（C95S,C141S,C188S and C195S）by site-directed mutagenesis of four cysteine residues of CU（Cys95,Cys141,Cys188 and Cys195）into serine.At the same time,we successfully optimized the fermentation process under the conditions of shaking table culture and 10 L fermentation tank,and the yield of CU and its mutants was up to 30 g·L^-1.The structure of uricase active tetramer was obtained by ammonium sulfate precipitation and xanthine affinity chromatography.Compared with CU,all of the four mutants retained more than 90%enzyme activity,which indicated that single mutation of Cys could not cause complete loss of enzyme activity.Circular dichroism indicated that the stability of theβ-barrels of the active tetramer protein was changed mainly by affecting the content ofβ-sheet.The results of thermostability and pH stability showed that the specific activity of C195S mutant was slightly lower than that of CU,but the stability of C195S mutant was significantly better than that of CU.The results showed that there was no necessary relationship between enzyme activity and stability of Cys mutant.In addition,we performed double mutation studies on nearby Cys188 and Cys195 and found that the active tetramer structure of the double mutant C188＆195S could not be obtained,suggesting that Cys188 affects hydrophobic interactions between subunits,the potential interaction between Cys188 and Cys195 was affected,and the formation or stability of the active tetramer was affected.In this thesis,the expression system of recombinant uricase in Escherichia coli was successfully constructed,the fermentation and purification process of recombinant uricase was established,and the Cys site of canine uricase in mammals was systematically studied.It was found that the Cys site had an important influence on the structure and function of canine uricase in mammals.It provides a useful reference for the structure confirmation and high stability screening of uricase in mammals.