A RECOMBINANT RIBOSOME-INACTIVATING PROTEIN FROM THE PLANT PHYTOLACCA-DIOICA L. PRODUCED FROM A SYNTHETIC GENE

Phytolacca dioica L. leaves produce at least two type-I ribosome-inact ivating proteins. Each polypeptide chain is subjected to different pos t-translational modifications giving rise to PD-L1 and PD-L2, and PD-L 3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and prelimin arily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generat ed by controlled oxidation of the reduced and denatured protein. The r ecombinant protein was indistinguishable from natural PD-L4 as isolate d from leaves of Phytolacca dioica, in both catalytic activity and pri mary structure. (C) 1998 Federation of European Biochemical Societies.