[Cloning and expression of urate oxidase and its application in serum uric acid analysis]

Anurate oxidase (uricase, EC 1.7.3.3) gene from Candida utilis AS2.117 was cloned by PCR amplification with primers derived from conserved regions of published uicase DNA sequence. The DNA sequence of cloned uricase gene was determined and a high homology compared to the reported gene was found. The cloned gene was inserted into Bam H I and Nde I sites of pET21a to create the recombinant plasmid pURO. In Escherichia coli BL21(DE3) host, the expression lever of uricase reached to about 40% of total soluble proteins of the cell. The western blot analysis confirmed the result of expression. Properties of the enzyme protein produced by E. coli BL21(DE3)/pURO were determined and similar with those of original protein from Candida utilis AS2.117. Furthermore, the thermostability of the expressed protein was enhanced. The purified recombinant uricase was used in serum uric acid analysis.