Recombinant Expression, Purification And Characterization Of Human Soluble Tumor Necrosis Factor Receptor 2

TNFR2 is aberrantly expressed on various cancer cells and highly immunosuppressive regulatory T cells (T-regs) accumulated in tumor microenvironment. As an oncoprotein and a stimulator of the immune checkpoint T-regs 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-regs 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-alpha with a K-D of 10.5 nM, and inhibited TNF-alpha-induced cytotoxicity in L929 cells with an EC50 of 0.57 mu g/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-alpha overload.