[Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase].
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.
Wu
Shuanglin
S
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
Chen
Bin
B
Liu
Chengqian
C
Ou
Yu
Y
Yi
Jianzhong
J
D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013552:Swine; D014503:Urate Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014503:Urate Oxidase