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TNFR2 is aberrantly expressed on various cancer cells and highly immunosuppressive regulatory T cells (T) accumulated in tumor microenvironment. As an oncoprotein and a stimulator of the immune checkpoint T that promote cancer cell survival and tumor growth, TNFR2 is considered to be a prospective target for cancer immunotherapy with the blockers developed to simultaneously inhibit abundant TNFR2 tumor-associated T and directly kill TNFR2-expressing tumors. The soluble ectodomain of TNFR2 has also been successfully applied in clinical treatment for TNF-related autoimmune diseases. Research practices on these therapeutic strategies need recombinant protein of human soluble TNFR2 (hsTNFR2); however, mass production of such biologics using eukaryotic cells is generally high-cost in culture materials and growth conditions. This study aimed to establish an efficient methodology to prepare bioactive hsTNFR2 through a prokaryotic expression system. Recombinant vector pMCSG7-hsTNFR2 was constructed and the His-tagged fusion protein expressed in E. coli was enriched in inclusion bodies. Recombinant hsTNFR2 was denatured, refolded, and then purified by affinity chromatography, tag removal, ion-exchange chromatography and gel filtration chromatography. A protein yield of 8.4 mg per liter of bacterial culture liquid with a purity of over 97% was obtained. Purified hsTNFR2 exhibited strong affinity to human TNF-α with a K of 10.5 nM, and inhibited TNF-α-induced cytotoxicity in L929 cells with an EC of 0.57 μg/ml. The biological activity assessed in vitro indicated that this soluble protein can be promisingly used in drug discovery for immunotherapy of TNFR2 cancers and treatment of autoimmune diseases featured by TNF-α overload.