Expression And Purification Of Onconase

Onconase, a member of RNase A superfamily, is a ribuonuclease, which is originally purified from oocytes and early embryo of North Leopard frog (Rana pipiens). It shows striking anti-tumor potential in vitro and in vivo.Anti-tumor effect of Onconase is directly related to its nucleic acid enzyme activity. Onconase can degradate cytoplasmic tRNA, which can activate caspase-dependent apoptosis pathway.Onconase is the first ribonuclease drug, which is now tested in Phase III clinical research for the treatment of malignant mesothelioma. With a unique structure and a small molecular weight, Onconase is highly stable. In clinical practice, it has less side effects, low immunogenicity and is not easy to produce resistance, etc.Therefore the research of Onconase on the academic and medical applications is significiant.We chose the E.coli expression system and Bac-To-Bac baculovirus expression system to prokaryotic and eukaryotic express Onconase protein respectively.In prokaryotic expression system, we used pMD18-T-Onconase, which was built by Invitrogen Corporation, as template. We inserted it into pET-30a(+) plasmid to construct pET-30a(+)-Onconase plasmid as the reconstructed expression plasmid. It was transformed into E.coli BL21(DE3) for the efficient expression of Onconase. The analysis using SDS-PAGE and Western blotting showed the specific protein band of approximate11KD. It indicated that the His-Onconase fusion protein had been successfully expressed in the E.coli BL21bacteria.In eukaryotic expression system, we amplified the full-length Onconasegene from pMD18-T-Onconase plasmid, and then we inserted it into plasmid pFastBacI of the Bac-To-Bac? Baculovirus expression system to form plasmid pFastBacI-Onconase.The recombinant shuttle vector named Baemid-Onconase was obtained from E.coli DH1OBac through transformation.After transfection of Baemid-Onconase with Cellfectin? II Reagent,recombinant Baculovirus(AcNPV-Onconase) was developed in Sf9cells.Then we used this viral stock to infect new Sf9cells togenerate His-Onconase titer baculoviral stock. After that, we used the baculoviral stock to infect Sf9cells for Onconase expression. At last, we harvested the cells at the different time points after infection (24,48,72hours). The specific11kD band was showed by SDS-PAGE and Westem blotting, which indicated that Onconase protein was expressed in Sf9cells. The expression level of Onconase protein arrived at the highest after infection with the recombinant virus AcNPV-Onconase for48hours.We used Ni-NTA agarose under denaturing conditions to purify the prokaryotic expressed His-Onconase fusion protein. After that the proteins were refolded through dialysis. The results of SDS-PAGE and Western blotting showed that His-Onconase fusion protein was obtained.We purified the eukaryotic expressed Onconase protein under native conditions using Ni-NTA agarose.The analysis using SDS-PAGE and Westem blotting showed that we obtained highly purified His-Onconase protein.In summary, using prokaryotic expression system and the Bac-To-Bac baculovirus eukaryotic expression system, we expressed and purified the Onconase protein.