Molecular Cloning And Characterization Of Pokeweed Antiviral Protein â… 

Objective: Pokeweed antiviral protein (PAP) is a 29kDa ribosome-inactivating protein (RIP), which attacks both eukaryotic (28s) and prokaryotic (23s) ribosome and remove a specific adenine base from the conserved region of the rRNA subunit. This prevents the binding of the elongation factor EF2, thus blocking protein sythesis. PAP has a strong cytotoxicity, inducing death or apoptosis of the cells. PAP had been proved to inhibit the replication of HTV-1 for many years. AU et al examined some plant RIPs including luffins for the ability to interfere with HIV-1 replication in a variety of mechanistic assays in vitro. In this article, we report that PAP I gene has been successfully cloned from the spring leaves of pokeweed and expressed in E. coli. The results suggested that the anti-HTV property of RIPs may be due to inhibition of HTV-1 integrase.To study the interaction between pokeweed antiviral protein and HTV-1 long terminal repeats (LTR) and the inhibition of integration.Methods: The cDNA sequence encoding PAP I was cloned from the fresh spring leaves of Phytolacca americana by RT-PCR. Nde I site was introduced at the upstream primer whereas a stop codon and Hind Ⅲ site were added at the downstream. The nucleotide sequences of the target DNA amplification fragment were sequenced after T-A cloning. The fragments digested with restriction endonucleases Nde I and Hind III were subsequently cloned into the vector pET-44a(+). E. coli BL21 (DE3)transformed with the expression plasmid was grown in LB at 37 ℃ to an optical density of 0.6. Expression was induced by adding IPTG to a final concentration of 1mmol/L and incubation with shaking at 37 ℃ for 3-5 h. The expression product was identified by 12% SDS-PAGE. The result showed that the recombinant product mainly existed in inclusion body. The recombinant protein in inclusion body was denatured with 8mol/L urea and was renatured by dialysis. The renatured proteins were affinitively purified on a Blue-Sepharose 6B column by elution with 1.0 mol/L sodium chloride. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity. The cytotoxicity of PAP I was evaluated by the MTT assay in HepG2 and A549 cells following fluid-phase endocytosis.Results: PAP has a significant toxicity on these cancer cells. PAP revealed a DNase activity on supercoiled DNA, and some divalent cations enhanced this activity apparently. The LTR binding assay has shown that PAP have a affinity to LTR in the presence of some divalent metal ions. PAP exhibited a suppressive effect on HTV-1 integrase in a dose-dependent manner. The final inhibition rate could reach to 80% in the ELISA-based assay. The conservtivity of the active site residues between PAP and MAP30 gave a possible explanation of integrase inhibition, since the latter one has been proven the significant inhibition activity on integration. Molecular docking have predicted the possible residues participated in the PAP/LTR interaction.Conclusion: The expression system of PAP-I has been successfully established. The inclusive bodies can be renatured by dialysis and the renatured PAP-I can bepurified by one-step affinity chromatography on the column of Blue-Sepharose 6B. The purity of final product was over 90 % analyzed by SDS-PAGE. The renatured protein shows cytotoxitic, which suggests that PAP-I can enter the cell byendocytosis. The result suggested that the inhibition activity on integration may correlate with the DGAL activity of PAP.