Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase.
- Author:
Jinlong ZHANG
1
;
Jun REN
;
Bing LI
;
Shuling LIU
;
Lihua HOU
;
Ling FU
;
Jianmin LI
;
Wei CHEN
Author Information
1. 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.
- Publication Type:Journal Article
- MeSH:
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
- From:
Chinese Journal of Biotechnology
2010;26(8):1102-1107
- CountryChina
- Language:Chinese
-
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.
