Cloning and molecular study of Aspergillus niger uric acid oxidase in Escherichia coli

Abstract

Urate oxidase enzyme or uricase (UOX) is a uric acid metabolizing enzyme that exists in most terrestrial animals, except primates (including human). An elevation in uric acid levels in the blood leads to many health problems, such as gout, electrolyte disturbances, and renal failure. Recently, recombinant UOX has been approved by the FDA to be used as a therapeutic agent for the treatment of hyperuricemia and Tumor Lysis Syndrome (TLS). Moreover, it is used to manufacture diagnostic kits for enzymatic uric acid determination. In the present study, we cloned the UOX gene from Aspergillus niger. The open reading frame of an UOX of 906 bp, encoding a protein of 302 amino acids, was synthesized using previous data from Genbank and cloned in pET-15b. The predicted amino acid sequence was used as a template to screen orthologues from evolutionarily related organisms and to explore their phylogenetic relationships. The monomeric subunit secondary structure was constructed; the molecular weight from the predicted amino acid sequences was 34.0 kDa and the isoelectric point value (pI) was7.19. The recombinant enzyme was expressed in soluble form as a 6-Histidine N-terminal fusion protein in E. coli BL21 (DE3) and purified using Ni-NTA-agarose affinity column. The UOX enzyme showed good activity toward uric acid.