Prokaryotic expression purification and identification of recombinant human soluble IL-6

To express and purify of recombinant human IL-6fromE.coli,and evaluate its activity using IL-6dependent cell line T1165.cDNA was generated from total RNA of activated T cells by RT-PCR and cloned into pET32a+.The resulting plasmid pET32a(+)/IL-6was transferred to E.coli BL21(DE3),and then overexpressed in E.coli cells by IPTG induction.Ni-NTA affinity chromatography was used for the purification.The purified protein was characterized and activity was validated by SDSPAGE and IL-6dependent cell line T1165.The recombinant human IL-6was soluble expressed in prokaryotes with high efficiency.SDS-PAGE analysis showed the molecular weight of the protein was 21kD.It can be identified by mouse anti-human IL-6specific antibody and the activity was 1×106 U/mg,determined by stimulating the proliferation of T1165cell.The present study provides a novel method to get soluble active human IL-6protein for further studying its biological activity.