title: [Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase].
abstract: 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.
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Laboratory of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Shuling
authors.initials: S
authors.lastname: Liu
authors.forename: Lihua
authors.initials: L
authors.lastname: Hou
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Jianmin
authors.initials: J
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D001231:Aspergillus flavus / Q000201:enzymology*; D003001:Cloning, Molecular; D003062:Codon / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014503:Urate Oxidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D014527:Uric Acid / Q000378:metabolism
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D014527:Uric Acid; D014503:Urate Oxidase