1. Graduated from MSc in Cellular and Molecular Biology of University of Science and Culture 2. microorganism bank.Iranian Biological Resource Center.ACECR.Tehran.Iran 3. microorganism bank.Iranian Biological Resource Center.ACECR.Tehran.Iran
Abstract
Introduction
Uricase, which is originated from a variety of organisms including bacteria, is an important agent for tracing of uric acid in blood and urine, and also is used in medicine to rectify purine metabolism disorders such as hyperuricemia and or gout. besides, uricase is employed in enzymatic detection of urate through pairing to 4aminoantyporine peroxidase.
Methods
In the present study a recombinant protein of uricase from streptomyces species was cloned, expressed and assessed. uricase coding sequence from the bacterial species was amplified by pcr and cloned in pet28a expression vector followed by plasmid sequencing to confirm the cloned gene. the recombinant plasmid was then transformed into e.coli bl21 (dl3). protein expression was performed at lower concentration of iptg and low temperature in order to achieve more soluble protein. affinity chromatography with ni-nta columns was used for purification of the extracted protein.
Results
Sds-page results indicated that the size of expressed protein is about 38kd and also confirmed the overexpression of the enzyme in e.coli expression system. although the expressed protein was seen in both soluble and insoluble forms, however, the proportion of soluble protein was considerably high according to the gel results. substantial amount of purified protein also showed how efficiently his tags at both sides of the protein help at column purification step. the specific activity of the purified uricase was about 10.1 u/ml/min.
Conclusion
High level expression and purification as well as extensive specific activity of the purified uricase make it as an acceptable candidate for treatment of purine metabolism deficiencies.
