Cloning and bacterial expression of monomeric short-chain dehydrogenase/reductase (carbonyl reductase) from CHO-K1 cells.
Mammalian carbonyl reductase (EC 1.1.1.184) is an enzyme that can catalyze the reduction of many carbonyl compounds, using NAD(P)H. We isolated a cDNA of carbonyl reductase (CHO-CR) from CHO-K1 cells which was 1208 bp long, including a poly(A) tail, and contained an 831-bp ORF. The deduced amino-acid sequence of 277 residues contained a typical motif for NADP+-binding (TGxxxGxG) and an SDR active site motif (S-Y-K). CHO-CR closely resembles mammalian carbonyl reductases with 71-73% identity. CHO-CR cDNA had the highest similarity to human CBR3 with 86% identity. Using the pET-28a expression vector, recombinant CHO-CR (rCHO-CR) was expressed in Escherichia coli BL21 (DE3) cells and purified with a Ni2+-affinity resin to homogeneity with a 35% yield. rCHO-CR had broad substrate specificity towards xenobiotic carbonyl compounds. RT-PCR of Chinese hamster tissues suggest that CHO-CR is highly expressed in kidney, testis, brain, heart, liver, uterus and ovary. Southern blotting analysis indicated the complexity of the Chinese hamster carbonyl reductase gene.
Terada
T
T
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Japan. tereda@phs.osaka-u.ac.jp
Sugihara
Y
Y
Nakamura
K
K
Sato
R
R
Inazu
N
N
Maeda
M
M
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D011994:Recombinant Proteins; D009249:NADP; D000429:Alcohol Oxidoreductases