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.
Li
Jianmin
J
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>
Chen
Zhao
Z
Hou
Lihua
L
Fan
Hongyan
H
Weng
Shaojie
S
Xu
Chun'e
C
Ren
Jun
J
Li
Bing
B
Chen
Wei
W
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