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Catalytically active soluble ecto‐5′‐nucleotidase purified after heterologous expression as a tool for drug screening
Author(s) -
Servos Jörg,
Reiländer Helmut,
Zimmermann Herbert
Publication year - 1998
Publication title -
drug development research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.582
H-Index - 60
eISSN - 1098-2299
pISSN - 0272-4391
DOI - 10.1002/(sici)1098-2299(199811/12)45:3/4<269::aid-ddr25>3.0.co;2-b
Subject(s) - recombinant dna , biochemistry , heterologous , fusion protein , 5' nucleotidase , biology , heterologous expression , microbiology and biotechnology , glutathione , enzyme , nucleotidase , gene
To establish a procedure for reproducible production of a soluble enzymatically active form of rat ecto‐5′‐nucleotidase, we heterologously expressed the polypeptide of the mature protein in insect cells. An expression construct was created consisting of this polypeptide and glutathione‐ S ‐transferase from Schistosoma japonicum as a fusion partner. Although infected insect cells did not secrete the enzyme into the medium, considerable amounts of recombinant protein were detected in the whole cell extract. A fraction of soluble protein yielding 5′‐nucleotidase activity could be purified from cells infected with recombinant baculovirus bearing the glutathione‐ S ‐transferase fusion construct. The catalytic properties of this recombinant protein correspond to those of the native protein isolated from animal tissues. Potential 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 in tissue culture experiments. Drug Dev. Res. 45:269–276, 1998. © 1998 Wiley‐Liss, Inc.