Expression, Refolding, Purification And Activity Of Mda-7/IL-24

Interleukin-24(IL-24), also known as melanoma differentiation associated gene-7(mda-7), can induce apoptosis in a wide variety of tumor cell types, whereas it has no toxicity in normal cells.In this study, we selected classic E.coli system, and successfully obtained the insoluble mda-7/IL-24inclusion bodies (IBs), it comprised up to25.8%of the total cellular protein.We optimized the mda-7/IL-24IBs denaturation and renaturation conditions. The result were20mM Tris,10mM EDTA,8M urea (pH9.5) as the denaturation buffer. Proposed a6times dialysis renaturation process method:decreasing the urea concentration (6M-3M-1.5M-0.75M-OM-OM), decreasing the pH (9.5-9.0-8.5-8.0-7.5-7.4),0.5-1.0kPa hydrostatic pressure, GSH:GSSG was10:1, decreasing the glycerin (20%-15%-10%-5%-0%-0%), and dissolved in PBS finally.We also optimized the fermentation conditions to obtain the IBs of improved structure, the result were:MgSO41.88g/L, CaCl21.84g/L, MnSO4·H2O0.50g/L, yeast extract5g/L, peptone10g/L and NaCl10g/L as the culture medium (pH7.0), cultured in37℃, induced with0.1mM IPTG in30℃when OD600reached0.6-0.8. It is confirmed by SEM that the IBs structure had been improved, which may be the direct reason why the denaturation rate increased. In general, the denaturation and renaturation rates increased by50.52%and83.78%, respectively.We also optimized the purification process of mda-7/IL-24protein. In the preliminary stage, we proposed a purification method which obtained the mda-7/IL-24protein of90%purity with purification after the refolding. Then, we proposed a simple purification method by washing IBs, thus it could eliminate the nickel affinity chromatography purification steps. We used buffer Ⅰ (20mM Tris-HCl,10mM EDTA,100mM NaCl,1.0%Triton X-100,2M Urea, pH8.5), buffer Ⅱ (20mM Tris-HCl,10mM EDTA,2.0%Triton X-100,4M Urea, pH8.5), buffer Ⅲ (20mM Tris-HCl,10mM EDTA,1.0%Triton X-100,4M Urea, pH9.5) and deionized water to wash the IBs respectively, each step IBs were washed for2h, then centrifugated at10,000rpm for30min. finally we obtained the mda-7/IL-24IBs protein of96%purity.The study confirmed the selective apoptosis of refolding mda-7/IL-24protein on A375and MCF-7by MTT and flow cytometry analysis.