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-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 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 preliminarily 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 generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.
Del Vecchio Blanco
F
F
Dipartimento di Chimica Organica e Biologica, Università Federico II di Napoli, Naples, Italy.
Cafaro
V
V
Di Maro
A
A
Scognamiglio
R
R
Siniscalco
G
G
Parente
A
A
Di Donato
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000235:genetics*
D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C105087:PD-S2 protein, Phytolacca dioica