title: High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase.
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 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.
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, People's Republic of China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Liu
authors.forename: Mingjie
authors.initials: M
authors.lastname: Wen
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Fangli
authors.initials: F
authors.lastname: Zhai
authors.forename: Jing
authors.initials: J
authors.lastname: Ruan
authors.forename: Liqing
authors.initials: L
authors.lastname: Zhang
authors.forename: Shentao
authors.initials: S
authors.lastname: Li
mesh_terms: D002175:Candida / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014503:Urate Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D014503:Urate Oxidase