Substitution And Recombinant Protein Expression Of Human Urate Oxidase Exon1-2

Urate oxidase (Uricase, EC1.7.3.3), a kind of oxidase of taking oxygen as a receptor, is widely distributed in the peroxisomes of mammalian liver cells. It could convert uric acid to a more soluble and easily excreted compound, i.e., allantoin. Urate oxidase has been found in the body of many species, while birds and some higher primates (eg apes and humans) lack functional urate oxidase in vivo and excrete uric acid as the end product of purine metabolism. If body produces too much uric acid and it could not excrete uric acid timely, or degration of excretion mechanism of uric acid in the body, all of them will affect the normal function of human cells. The highly concentration uric acid for a long time in the body will lead to gout.Currently, drugs for reducing uric acid levels in the body are divided into three categories: drugs for promoting excretion of uric acid, drugs for inhibition synthetic of uric acid and urate oxidase. Studies have shown that urate oxidase can far superior in reducing uric acid levels than other drugs in scale and speed. Since the urate oxidase in human body does not have activity, the current clinical application of urate oxidase is mainly rooted from Aspergillus flavus. Since it is a foreign protein with immunogenicity, clinical application is greatly limited. It has been reported that the urate oxidase with PEG-modified can reduce its immunogenicity, but enzyme activity has decreased greatly after PEG modified. Uricase gene mutations of human may result in the formation of pseudogenes. If we are enable to make the urate oxidase “resurrection”, it must be reduce the immunogenicity of the enzyme to a large extent.To further explore the reason of inactivated human urate oxidase and to search for urate oxidase from mammalian sources with more closely human homology at the same time, in this study, we chose method of bioinformatics, designed some forward and backward primers according to sequence published under the NCBI, and then extracted total RNA from porcine liver orgization. Porcine uricase gene is obtained by RT-PCR. Cloning pUOX (porcine urate oxidase)is inserted into the prokaryotic expression vector pET-22b(+) to construct the recombinant plasmid pET-22b/pUOX, and then transformed into Escherichia coli BL21(DE3)expression bacterial strain to realize efficient expression. SDS-PAGE electrophoresis with12%separated gel to detect protein expression and examining engineering strain of porcine uricase by UV absorption, the protease activity of pET-22b/pUOX is1.962U/mL. The gene structure was confirmed by sequence analysis. The human uricase gene is extracted by this laboratory from human liver organization, and preserved in BL21(DE3) expression strain. A clear protein band can be detected by SDS-PAGE protein electrophoresis, but non-protein enzyme activity. Exon2and5in the human uricase have existed the the codon CGA coding for arginine mutate to terminal codon TGA, and research indicates that the mutation of exon2is the beginning of evolution. So, we replace corresponding exon1and2of porcine and human urate oxidaseene so as to construct human-porcine recombinant uricase gene for expression. And then induced by IPTG, the recombinant protein is checked by SDS-PAGE electrophoresis with12%separation gel. which shows a clear protein band about34kDa, this result is consistent with the expected molecular weight of303amino acids. Determination of enzyme activity of the human-porcine recombinant protein is0.833U/mL. The gene structure was confirmed by sequence analysis. Compared with porcine uricase, the enzyme activity of human-porcine uricase recombinant engineering bacteria pEU-H1-2has apparently decreased by BlastN alignment online, which has existed six sense mutations. The successful construction of pEU-H1-2recombinant engineering bacteria with activity has proved that the exon1-2of human urate oxidase is not the main reason to induce inactivation. The successful construction of human-pig engineering bacteria has laid a good foundation to inactivation studies of human urate oxidase gene, it also provide a useful following experiments.