Abstract
The cDNA for porcine kidney D-amino acid oxidase (DAO) was cloned by means of the reverse transcription-polymerase chain reaction system from porcine kidney RNA and over-expressed in Escherichia coli which had been transformed with a vector containing the DAO cDNA. The expressed DAO was purified to homogeneity by a three-step procedure, i.e., heat-treatment, DEAE Sepharose column chromatography, and hydroxyapatite column chromatography. The purified DAO preparation, rDAO (recombinant DAO), showed an identical UV-visible absorption spectrum and catalytic activity with those of the wild-type enzyme purified from porcine kidney. Crystallization of rDAO was performed by the hanging-drop method and crystals of suitable quality for X-ray crystallography were obtained. The crystals so obtained diffracted to 2.5 A with a conventional X-ray source, and to 2.0 A with synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 110.3, b = 92.9, c = 71.6 A. A Vm value of 2.35 A3/Da indicates that there are two subunits related by a twofold non-crystallographic axis in the asymmetric unit. Two heavy atom derivatives have been identified.
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