Bovine cytosolic IMP/GMP-specific 5′-nucleotidase: cloning and expression of active enzyme in Escherichia coli

Abstract

A cDNA coding for bovine cytosolic IMP/GMP-specific 5ʹ-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5ʹ and 3ʹ rapid amplification of cDNA ends protocols (5ʹ and 3ʹ RACE). Two products were isolated: a 5ʹ RACE 1.6 kb fragment and a 3ʹ RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5ʹ-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5ʹ-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5ʹ-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5ʹ-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5ʹnucleotidase has been described.