Bovine cytosolic IMP/GMP-specific 5'-nucleotidase: cloning and expression of active enzyme in Escherichia coli.
A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE). Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' 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 5'-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5'-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 5'-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 5'-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 5'nucleotidase has been described.
Allegrini
S
S
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Science, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden.
Pesi
R
R
Tozzi
M G
MG
Fiol
C J
CJ
Johnson
R B
RB
Eriksson
S
S
D015720:5'-Nucleotidase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006157:Guanosine Monophosphate / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D007291:Inosine Monophosphate; D006157:Guanosine Monophosphate; D015720:5'-Nucleotidase