Molecular Cloning and Expression of Human Liver Biliverdin-IX[beta] Reductase

The cDNA encoding for human liver isozyme I of biliverdin-IXβ reductase was cloned from human liver cDNA libraries. The constructed cDNA of 853 bp in length contained an entire reading frame coding 206 amino acid residues. It was found that isozyme I of biliverdin-IXβ reductase is identical to human erythrocyte NADPH-flavin reductase recently reported in a communication by Chikuba et al., Biochem. Biophys. Res. Commun., 198, 1170-1176 (1994). We, further, characterized this mRNA by Northern blot analyses of poly(A) RNA from four different human fetal tissues and eight different human adult tissues showed hybridization mainly to liver RNA in fetal tissues and to skeletal muscle and liver RNA in adult tissues. Sourthern blot analysis indicated that isozyme I of biliverdin-IXβ reductase appeared to be a single copy gene. Insertion of the enzyme-coding sequence into an expression vector pET-3c yielded relatively high amounts of the active enzyme in E. coli. The amino terminal sequence of the recombinant protein was identical to that of native enzyme, indicating that E. coli also removed the N-terminal methionine to produce the mature form. The recombinant and native biliverdin-IXβ reductases were indistinguishable as far as their mobility on SDS-PAGE gel, immunoreactivity and specific activity were concerned.