Catalytically active soluble ecto-5 '-nucleotidase purified after heterologous expression as a tool for drug screening

To establish a procedure for reproducible production of a soluble enzymatic ally active form of rat ecto-5'-nucleotidase, we heterologously expressed t he polypeptide of the mature protein in insect cells. An expression constru ct was created consisting of this polypeptide and glutathione-S-transferase from Schistosoma japonicum as a fusion partner. Although infected insect c ells did not secrete the enzyme into the medium, considerable amounts of re combinant protein were detected in the whole cell extract. A fraction of so luble protein yielding 5'-nucleotidase activity could be purified from cell s infected with recombinant baculovirus bearing the glutathione-S-transfera se fusion construct. The catalytic properties of this recombinant protein c orrespond to those of the native protein isolated from animal tissues. Pote ntial agonists and inhibitors of P2 receptors can function as inhibitors of ecto-5'-nucleotidase. The glutathione-S-transferase fusion protein may be used in experiments for drug screening, for the study of the interaction of immobilized ecto-5'-nucleotidase with other proteins, or for application i n tissue culture experiments. Drug Dev. Res. 45:269-276, 1998. (C) 1998 Wil ey-Liss, Inc.