High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli |
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Authors: | Li Jianmin Chen Zhao Hou Lihua Fan Hongyan Weng Shaojie Xu Chun'e Ren Jun Li Bing Chen Wei |
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Institution: | 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 |
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Abstract: | 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. |
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