Overexpression of pokeweed antiviral protein in Escherichia coli: purification, characterization and comparison with native sources

Abstract

Pokeweed antiviral proteins (PAP, PAP-II, PAP-S) represent a family of ribosome inactivating proteins (RIPs) isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). They not only inhibit the mechanical transmission of plant viruses but also inactivate both eukaryotic and prokaryotic protein synthesis. One of them, PAP, was expressed with the help of the highly inducible prokaryotic expression vector pOPE 90 in Escherichia coli and the biological activity of the recombinant PAP (rPAP) was compared with the PAP purified from spring leaves of Phytolacca americana. After extraction and solubilization, rPAP was highly purified by anion and cation exchange chromatography. Immunoblotting with antibodies revealed identical immunological reactivity between recombinant or native PAP. In a rabbit reticulocyte translation system, rPAP was more efficient than native PAP to inhibit protein synthesis. The values of IC 50 from native PAP and recombinant PAP were determined as 5.10 −11 M and 5.10 −12 M, respectively. No difference was observed when the toxicity of native and rPAP was tested on established cell lines. PAP can be expressed in equivalent active form in Escherichia coli and its properties were thus similar to those of the native protein. Post-translational modifications of PAP in pokeweed did not seem to alter its ability to inhibit protein synthesis.