Expression of Mature Pokeweed Antiviral Protein with or without C-Terminal Extrapeptide in Escherichia coli as a Fusion with Maltose-Binding Protein

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  Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAP_MC and PAP_M), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAP_MC and rPAP_M) were successfully expressed in E. coli at 25°C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAP_MC) and 5.5 mg/liter (rPAP_M). rPAP_M was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAP_MC and rPAP_M were as active as the native PAP_M from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAP_MC on both ribosomes were decreased at least 60-fold by fusion with MBP.