Expression of normal and abnormal porcine kidney D-amino acid oxidase in Escherichia coli: purification and characterization of the enzymes.
Expression plasmids for normal and abnormal porcine D-amino acid oxidases (E.C. 1.4.3.3, DAO) have been constructed from cloned cDNA that encodes the entire protein sequence of DAO, and the enzymes were expressed in Escherichia coli cells on a large scale. The expressed enzymes were purified to apparent homogeneity. The molecular weight of the normal DAO (38 kD) was identical with that of DAO purified from porcine kidney, whereas that of the abnormal DAO was 39 kD, which comprised the normal DAO with an additional decapeptide at its amino terminus. However, the specific activities of the two enzymes were comparable with that of natural DAO. The results indicate that the bulky decapeptide does not affect the structure necessary for the catalytic function of DAO in the amino-terminal region. The use of a GTG triplet in the 5'-untranslated region of DAO cDNA as the initiation codon for the synthesis of the abnormal DAO is suggested.
Watanabe
F
F
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan.
Fukui
K
K
Momoi
K
K
Miyake
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine
D003062:Codon; D011994:Recombinant Proteins; D004247:DNA; D003605:D-Amino-Acid Oxidase