Cloning and expression of ecto 5 '-nucleotidase from the cattle tick Boophilus microplus

Although 5'-nucleotidases are ubiquitous in higher vertebrates, the arthrop od enzymes have been little studied, The cDNA sequence of the mature 5'-nuc leotidase from the tick Boophilus microplus was therefore determined (GENBA NK accession number: U80634), The enzyme has 39-41% sequence identity with the vertebrate 5'-nucleotidases and contains binuclear metal ion binding si tes. There are no significant introns within the coding region of the genom ic sequence, Southern blot analysis indicates the presence of multiple rela ted genes encoding 5'-nucleotidases, Recombinant tick 5'-nucleotidase was e xpressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in ab undance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This rep resents the first successful expression and characterization of enzymatical ly active recombinant 5'-nucleotidase from any organism. Supplementation of the culture medium with 25 mu M zinc resulted in a twofold increase in the activity of the expressed protein. The enzyme was purified to homogeneity. It exists under non-denaturing conditions as a homodimer, with an apparent molecular mass of 135 kDa. The K-m for the hydrolysis of AMP was 0.37 mu M and the k(cat) = 11.5/s, in agreement with data for the native enzyme.