High-level expression and purification of biologically active recombinant pokeweed antiviral protein

Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phy tolacca americana, is a naturally occurring single-chain ribosome-inactivat ing protein, which catalytically inactivates both prokaryotic and eukaryoti c ribosomes. The therapeutic potential of PAP has gained considerable inter est in recent years due to the clinical use of native PAP as the active moi ety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quanti ties of a homogenously pure and active PAP preparation with minimal batch t o batch variability from its natural source, Previous methods for expressio n of recombinant PAP in yeast, transgenic plants and Escherichia coil have resulted in either unacceptably low yields or were too toxic to the host sy stem. Here, we report a successful strategy which allows high level express ion of PAP as inclusion bodies in E. coil. Purification of refolded recombi nant protein from solubilized inclusion bodies by size-exclusion chromatogr aphy yielded biologically active recombinant PAP (final yield: 10 to 12 mg/ L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain s ufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active r ecombinant PAP from E. coil. (C) 1999 Academic Press.