cDNA cloning and expression of pokeweed antiviral protein from seeds in Escherichia coli and its inhibition of protein synthesis in vitro.
Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). We isolated, sequenced and characterized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds. The cDNA of PAP-S consists of 1249 nucleotides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%). It is known from literature that PAP-S is more active in inhibiting protein synthesis than other members of the PAP family. Therefore, the cDNA of PAP-S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified from spring leaves. In a rabbit translation system, the median inhibitory concentrations (IC50) of recombinant PAP-S and native PAP were determined as 0.07 and 0.29 nM, respectively. Although the PAP-S protein in seeds is glycosylated, PAP-S can be expressed in Escherichia coli in a very active form, indicating that post-translational modification in pokeweed does not seem to alter its ability to inhibit protein synthesis.
Poyet
J L
JL
Laboratoire de Biochimie et de Biologie Moléculaire, UFR des Sciences et des Techniques, Besancon, France.
Hoeveler
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1; D012639:Seeds / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein