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cDNA cloning and expression of pokeweed antiviral protein from seeds in Escherichia coli and its inhibition of protein synthesis in vitro
Author(s) -
Poyet Jean-Luc,
Hoeveler Arnd
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00250-0
Subject(s) - escherichia coli , complementary dna , biology , recombinant dna , microbiology and biotechnology , antiviral protein , amino acid , peptide sequence , protein a/g , biochemistry , protein biosynthesis , gene , rna , fusion protein
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 (IC 50 ) 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.