Purification and Characterization of the Mda-7 Tumor Suppressor Protein Expressed in Insect Cells

The melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24) is a novel cytokine, which exhibits both tumor suppressing and immunostimulatory activities. Mda-7 has the ability to restrain growth and induce programmed cell death (apoptosis) in a wide variety of human cancer cells without having similar effects on normal cells. This study was performed to investigate the proteochemical and tumor suppressing properties of soluble human mda-7 produced in baculovirus-insect cell expression vector system. The effects of the post-translational modifications, especially glycosylation, on the activity of mda-7, are controversial. To investigate the role of glycosylation, a polyhistidine tagged mda-7 (mda-7-His) fusion protein was produced in two different insect cell lines: in Spodoptera frugiperda 9 (Sf9) and in Mimic cells, the latter cell line has been genetically engineered to stably express a variety of mammalian glycosyltransferases allowing processing of complex N-glycans to a similar extent with mammalian cells. Furthermore, the production and purification protocols of the proteins were optimized and the proteochemical properties of the fusion proteins were characterized by SDS-PAGE and Western blotting. Finally, the induction of apoptosis by the mda-7-His fusion protein was tested in human MDA-MB-435 breast carcinoma and PC-3 prostate adenocarcinoma cells by Trypan blue staining and Annexin V labeling. The fusion proteins were successfully produced and purified from insect cells. Analyzed by Western blotting, the mda-7-His expressed in Sf9 cells was approximately 23 kDa in size, representing the non-glycosylated form of the protein. The protein sample from the Mimic cells had two main bands, about 25 kDa and 45 kDa in size, representing the non-glycosylated or partially glycosylated and fully glycosylated forms of the protein, respectively. Despite the differences in glycosylation between the proteins, both of the proteins retained their apoptosis inducing functions. Accordingly, with PC-3 cell line the percentage of apoptotic cells was up to 26% with mda-7-His produced in Sf9 cells and 25% with the mda-7-His produced in Mimic cells. From these result it can be concluded that both glycosylated and non-glycosylated soluble mda-7-His produced in baculovirus-insect cell expression system are able to induce apoptosis in human cancer cells. These results demonstrate that this expression system can be employed to easily produce large amounts of biologically active mda-7, rendering it useful for further studies on the structure and function of mda7.