Crystallization of Expressed Porcine Kidney D-Amino Acid Oxidase and Preliminary X-Ray Crystallographic Characterization

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 Å with a conventional X-ray source, and to 2.0 Å with synchrotron radiation. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unitcell dimensions of a = 110.3, b = 92.9, c=71.6 Å. A V_m 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.