Preliminary Study On The Renaturation Of The PTD4-VP3 Fusion Protein Inclusion Bodies

VP3 protein (also known as Apoptin) is a small molecule protein derived from chicken anemia virus (chicken anemia virus, CAV). Research has shown that, apoptin can selectively induced transformed cells and tumor cells to apoptosis, but had no effect on normal diploid cells, which was considered an ideal anti-cancer drug. Previous study in our laboratory had successfully constructed PTD4-VP3 fusion protein expression vector through the prokaryotic expression system of E. coli, and we obtained PTD4-VP3 fusion protein after induced expression. But the experiment showed that large amounts of the protein were inactive existing in the form of inclusion bodies. The soluble expression is not high. In this study, we aim to renature the inclusion bodies of PTD4-VP3 fusion protein, then detect its biological activity by MTT assay and flow cytometry, so as to establish a method of large scale preparation.Expression strain E.coliBL21(DE3)PlysS containing recombinan plasmid pET-28a-PTD4-VP3 was inoculated in LB medium containing 50μg/ml kanamycin to induce the expression of PTD4-VP3 fusion protein. Then the fusion protein inclusion bodies were dissolved in 8M urea overnight and purified them by affinity chromatography. The purified inclusion bodies was dialyzed in the concentration gradient of urea to renature. Then the purity of renatured inclusion bodies were detected by SDS-PAGE and the final yield was detected by BCA protein quantitation. And the effect of inhibiting tumor cell proliferation of renatured inclusion bodies was detected by MTT assay, and apoptosis rate of renatured inclusion bodies to tumor cells was observed by flow cytometry.The result of SDS-PAGE showed PTD4-VP3 expressed by E.coli majorly existed in the form of inclusion bodies and the soluble fusion protein expressed was little. BCA protein quatitation showed up to 8.5±0.7mg PTD4-VP3 inclusion bodies can be obtained from 1 L E.coli culture, but only 0.7±0.3mg PTD4-VP3 soluble protein can be obtained. And MTT assay showed that on the protein concentration of 25μg/ml, 50μg/ml, 75μg/ml and 100μg/ml respectively, the rate of inhibiting tumor cell proliferation of soluble PTD4-VP3 fusion protein were 20±1.7%, 32±1.9%, 50±2.5% and 58±2.8% respectively, while the renatured inclusion bodies were 40±2.1%, 70±2.2%, 71±2.4% and 91±2.9% respectively. The result of flow cytometry showed that on the protein concentration of 5μg/ml, 10μg/ml, 20μg/ml, 25μg/ml respectively, the rate of Hela cells apoptosis induced by soluble expression were 41.8±2.2%,60.3±3.2%,64.6±3.8%,59.8±4.5% respectively, while the rate of Hela cells apoptosis induced by renatured PTD4-VP3 inclusion bodies were 40.5±2,8%,63.1±3.6%,62.3±4.5%,61.4±3.8%. The PTD4-VP3 inclusion bodies were successfully renatured and the method of large scale preparation of PTD4-VP3 was established. .