Characterization of a soluble IL-6 receptor α mutant C277D/H280I expressed in E. Coli

Abstract

The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor α(IL-6Rα) and the signal transducer gp130. To construct the antagonist of IL-6, a DNA segment encoding the soluble human lL-6Rα(shlL-6Rα) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold. DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells. DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.