Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, People's Republic of China.
Appl Microbiol Biotechnol. 2011 Nov;92(3):529-37. doi: 10.1007/s00253-011-3244-0. Epub 2011 May 15.
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 P2(1)2(1)2(1) 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.
我们成功地在大肠杆菌中实现了产朊假丝酵母尿酸酶的高效表达，并建立了一种有效的三步式蛋白纯化方案。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基质辅助激光解吸/电离飞行时间质谱分析，确认重组蛋白的纯度大于 98%，且比活为 38.4IU/mg。产朊假丝酵母尿酸酶的晶体在 18°C 下使用 25%的聚乙二醇 3350 作为沉淀剂生长。晶体的衍射分辨率达到 1.93Å，晶体属于空间群 P2(1)2(1)2(1)，其晶胞参数为 a = 69.16Å，b = 139.31Å，c = 256.33Å，α = β = γ = 90°。产朊假丝酵母尿酸酶的晶体结构与蛋白数据库中其他物种同源尿酸酶的报道结构高度相似，表明蛋白质的三维结构对催化活性至关重要。
