Optimization of effective factors in the expression of recombinant urate oxidase enzyme using response surface methodology

The aim of the present study is to optimize the effective factors in the expressions and purifications process of uricase (urate oxidase) from E. coli. Urate oxidase is a class of oxidoreductase enzymes that lacks cofactor and converts the uric acid to allantoin. The purified uricase can be used for treating gout disease and hyperuricemia. Also, uricase is applied as a reagent in clinical diagnostic kits to specify the concentration of uric acid in the blood. Response surface methodology (RSM) has been widely applied as an effective technique to demonstrate the relationships and interactions among multiple variables by reducing the number of experimental trials due to the less laborious and time-consuming nature of the method. Based on the amount of IPTG concentration, temperature and incubation time that were the effective factors in expression process, the central composite design (CCD) was carried out and ۱۷ runs were designed using the Design-Expert software version ۱۱. In this study, recombinant urate oxidase enzyme from Escherichia coli BL۲۱ was expressed in ۱۷ runs. The protein was then purified using affinity chromatography and determined protein concentration by bradford method. The quadratic equation was used to describe the response of the system (UOX concentration). The optimized conditions were obtained, including the ۰.۵۵ mM concentration of IPTG and expression temperature ۳۲.۲۵ °C and ۱۹ h incubation time. The results were corroborated by Bradford method that was showed ۰.۴۲ mg/ml uricase in the best condition and furthermore analysis of protein Gels (SDS-PAGE) by molecular mass of ۳۴ kDa.

کلیدواژه ها:

Response surface methodology ، Uricase ، IPTG