Mammalian 5 '(3 ')-deoxyribonucleotidase, cDNA cloning, and overexpressionof the enzyme in Escherichia coli and mammalian cells

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whos e physiological function is not known. It was earlier purified to homogenei ty from human placenta. We determined the amino acid sequences of several i nternal peptides and with their aid found an expressed sequence tag clone w ith the complete cDNA for a murine enzyme of 23.9 kDa, The DNA was cloned i nto appropriate plasmids and introduced into Escherichia coli and ecdyson-i nducible 293 and V79 cells. The recombinant enzyme was purified to homogene ity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells s howed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked th e 5'-end corresponding to 51 amino acids of the murine enzyme. Several poly merase chain reaction-based approaches to find this sequence met with no su ccess. A mouse/human hybrid cDNA that had substituted the missing human 5'- end with the corresponding mouse sequence coded for a fully active enzyme.