Abstract
Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-395) contains supplementary material, which is available to authorized users.
Highlights
Urate oxidase (EC 1.7.3.3) plays an important role in purine degradation pathway and catalyzes uric acid oxidation into allantoin, H2O2 and CO2 in the presence of oxygen (Collings et al 2010)
Uric acid is considered to donate more than half the antioxidant capacity of blood resulting in diminution of age-specific cancer and enhancement of life expectancy, but hyperuricemia due to tumor lysis syndrome, excessive dietary purine intake and genetic basis leads to
Preparation of urate oxidase expression cassette The expression construct was prepared in order to provide a high level expression of urate oxidase in P. pastoris
Summary
Urate oxidase (EC 1.7.3.3) plays an important role in purine degradation pathway and catalyzes uric acid oxidation into allantoin, H2O2 and CO2 in the presence of oxygen (Collings et al 2010). The Aspergillus flavus urate oxidase (135 kDa) contains four identical subunits, in which each subunit is associated with one active site. Urate oxidase is a non-glycosylated enzyme having no intra- or inter-disulfide bonds with a blocking acetyl group located at the N-terminal (Legoux et al 1992). Urate oxidase is not expressed in human and other higher primates. Uric acid is considered to donate more than half the antioxidant capacity of blood resulting in diminution of age-specific cancer and enhancement of life expectancy, but hyperuricemia due to tumor lysis syndrome, excessive dietary purine intake and genetic basis leads to
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