ENGINEERING OF THERAPEUTIC ASPERGILLUS FLAVUS URICASE USING SITE-DIRECTED MUTAGENESIS

As a therapeutic enzyme, Aspergillus flavus (uricase or; EC 1.7.3.3), is used for treatment of urate deposits, gout and nephropathy, hyperuricemia and tumor lysis syndrom (TLS). Despite desirable kinetic features, fragile stability of uricase limits its wide range applications. Therefore, several approaches have developed such as protein engineering and genetic manipulations to overcome these problems. The main aim of the current research was the design and creation of disulfide bridge in therapeutic enzyme uricase for the first time.
By the help of bioinformatics studies and based on protein data bank (PDB) code: 1xxj, the potential points for disulfide bridge formation were selected. To create mutations, site-directed mutagenesis using SOE-PCR was employed.
According to computational tools, six potential pairs including three intra and three inter-chains were predicted to form disulfide when mutated to cysteins as followings: Ser145-Thr185, Ala235-Val248, His256-Gln281 and Ala6-Cys290, Ser282–Ser282, Asn12-Asp283. By means of SOE-PCR, mutation and cloning of Ala6 and Ser282 was implemented and verified by colony PCR, digestion check and eventually by sequencing.
In the current research, we successfully determined the location for mutating to cystein and fortunately implemented mutagenesis by SOE-PCR.