Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm . Therefore PAP is a good candidate to be used as an immunotoxin. We constr ucted a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites o n several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies, After purificatio n under denaturing conditions, renaturated GnRH-PAP shows an IC50 of 3 nM o n in vitro translation assays and selectively inhibits the growth of the Gn RH receptor positive Ishikawa cell line (ID50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect. (C) 2000 Federation of European Biochemical Societies.