Urate oxidase (Uox) plays a pivotal role in uric acid (UA) degradation, and it has been applied in controlling serum UA level in clinical treatment of hyperuricemia (HUA). However, because Uox is a heterogenous protein to the human body, the immune rejections typically occur after intravenous administration, which greatly hampers the application of Uox-based agents. In this study, we used Lactococcus lactis NZ9000, a food-grade bacterium, as a host to express exogenous Uox genes, to generate the Uox-expressing engineered strains to treat HUA. Aspergillus flavus-derived Uox (aUox) and the "resurrected" human-derived Uox (hUox) were cloned into vector and expressed in NZ9000, to generate engineered strains, respectively. The engineered NZ9000 strains were confirmed to express Uox and showed UA-lowering activity in a time-dependent manner in vitro. Next, in an HUA mice model established by oral gavage of yeast paste, the UA levels were increased by 85.4% and 106.2% at day 7 and day 14. By contrast, in mice fed with NZ9000-aUox, the UA levels were increased by 39.5% and 48.3% while in mice fed with NZ9000-hUox were increased by 57.0% and 82.9%, suggesting a UA-lowering activity of both engineered strains. Furthermore, compared with allopurinol, the first-line agent for HUA treatment, mice fed with NZ9000-aUox exhibited comparable liver safety but better kidney safety than allopurinol, indicating that the use of engineered NZ9000 strains not only alleviated kidney injury caused by HUA, but could also avoided the risk of kidney injury elicited by using allopurinol. Collectively, our study offers an effective and safe therapeutic approach for HUA long-term treatment and controlling.
Animals ; Lactococcus lactis/metabolism* ; Urate Oxidase/genetics* ; Mice ; Uric Acid/blood* ; Hyperuricemia ; Humans ; Administration, Oral ; Aspergillus flavus/genetics* ; Male

In order to characterize a thermostable urate oxidase (Uox) from Microbacterium sp. strain ZZJ4-1, we cloned its gene (uox). The open reading frame of uox contained 894 base pairs and encoded a protein with 297 amino acids. Alignment of gene sequences indicated there was no obvious identity with the most reported uox and that 72% identity was found with uox from Arthrobacter globiformis. We inserted the gene into the plasmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 (DE3). After the purification of the recombinant Uox by the HisBind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. It was stable below 65 degrees C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag+, Zn2+, CU2+ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.
Actinomycetales ; enzymology ; genetics ; Amino Acid Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Urate Oxidase ; genetics ; metabolism

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.
Aspergillus flavus ; enzymology ; Cloning, Molecular ; Codon ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification ; Uric Acid ; metabolism

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.
Animals ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Swine ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification