High-yield expression,purification, characterization,and structure determination of tag-free <Emphasis Type="Italic">Candida utilis</Emphasis> uricase |
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Authors: | Xiaojuan Liu Mingjie Wen Jing Li Fangli Zhai Jing Ruan Liqing Zhang Shentao Li |
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Institution: | (1) Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, People’s Republic of China;(2) Department of Chemistry and Molecular Biology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, People’s Republic of China;(3) College of Life Science, Capital Normal University, Beijing, 100048, People’s Republic of China; |
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Abstract: | We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P212121 with unit cell parameters a?=?69.16 Å, b?=?139.31 Å, c?=?256.33 Å, and α?=?β?=?γ?=?90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities. |
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