Cloning and expression of ecto 5-nucleotidase from the cattle tick Boophilus microplus.
Although 5'-nucleotidases are ubiquitous in higher vertebrates, the arthropod enzymes have been little studied. The cDNA sequence of the mature 5'-nucleotidase from the tick Boophilus microplus was therefore determined (GENBANK accession number: U80634). The enzyme has 39-41% sequence identity with the vertebrate 5'-nucleotidases and contains binuclear metal ion binding sites. There are no significant introns within the coding region of the genomic sequence. Southern blot analysis indicates the presence of multiple related genes encoding 5'-nucleotidases. Recombinant tick 5'-nucleotidase was expressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in abundance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This represents the first successful expression and characterization of enzymatically active recombinant 5'-nucleotidase from any organism. Supplementation of the culture medium with 25 microM 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 Km for the hydrolysis of AMP was 0.37 microM and the k(cat) = 11.5/s, in agreement with data for the native enzyme.
Liyou
N
N
CSIRO Tropical Agriculture, Long Pocket Laboratories, Indooroopilly, Queensland, Australia.
Hamilton
S
S
Elvin
C
C
Willadsen
P
P
D015720:5'-Nucleotidase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013987:Ticks / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D015720:5'-Nucleotidase