High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli.
The entire encoding region for Aspergillus flavus uricase was cloned into pET-32a and expressed in Escherichia coli BL21 (DE3). The uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form. A scalable process aimed to produce and purify multi-gram quantities of highly pure, recombinant urate oxidase (rUox) from E. coli was developed. The rUox protein was produced in a 30 L fermentor containing 25 L of 2x YT medium and purified to >99% purity using hydrophobic interaction, anion-exchange, and gel filtered chromatography. The final yield of purified rUox from fermentation resulted in approximately 27 g of highly pure, biologically active rUox per kg of cell paste (approximately 238 mg/8.8 g cell paste/L). The results presented here exhibit the ability to generate multi-gram quantities of rUox from E. coli that may be used for the development of pharmaceutics of reducing the hyperuricemia.
State Key Laboratory of Pathogens and Biosecurity, Laboratory of Applied Molecular Biology, Beijing Institute of Microbiology and Epidemiology, 20 FengTai Dongdajie Street, Beijing 100071, PR China. ljmqz@yahoo.com.cn <ljmqz@yahoo.com.cn>
Jianmin
J
Li
Zhao
Z
Chen
Lihua
L
Hou
Hongyan
H
Fan
Shaojie
S
Weng
Chun'e
C
Xu
Jun
J
Ren
Bing
B
Li
Wei
W
Chen
D001231:Aspergillus flavus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014503:Urate Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D014503:Urate Oxidase