Crystallization of expressed porcine kidney D-amino acid oxidase and preliminary X-ray crystallographic characterization.
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.
Setoyama
C
C
Department of Biochemistry, Kumamoto University School of Medicine.
Miura
R
R
Nishina
Y
Y
Shiga
K
K
Mizutani
H
H
Miyahara
I
I
Hirotsu
K
K
D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D013552:Swine; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase