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, i ncluding a poly(A) tail, and contained an 831-bp ORF. The deduced amino-aci d sequence of 277 residues contained a typical motif for NADP(+)-binding (T GxxxGxG) and an SDR active site motif (S-Y-K). CHO-CR closely resembles mam malian carbonyl reductases with 71-73% identity. CHO-CR cDNA had the highes t 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 carbo nyl compounds. RT-PCR of Chinese hamster tissues suggest that CHO-CR is hig hly expressed in kidney, testis, brain, heart, liver, uterus and ovary. Sou thern blotting analysis indicated the complexity of the Chinese hamster car bonyl reductase gene.