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 = ß = 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.
Subject(s)
Candida/enzymology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Urate Oxidase/chemistry , Urate Oxidase/metabolism , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Gene Expression , Models, Molecular , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Urate Oxidase/genetics , Urate Oxidase/isolation & purificationABSTRACT
Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS-PAGE and MALDI-TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1â Å resolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.25, b = 63.29, c = 131.81â Å.