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.