High level expression of non-fusion recombinant human IL-18 in E. coli

Objective To construct a expression vector which can produce a high level expression of mature human IL 18 (mhIL 18) in E coli . Methods The cDNA encoding mhIL 18 was amplified from human Kupffer cells with RT PCR. The recombinant expression plasmid petTT/mhIL 18 was constructed by cloning cDNA of mhIL 18 into petTT vector and then transformed to E. coli DH5α. IPTG was added into the E. coli culture to express the recombinant mhIL 18. After the harvested bacteriawere dispersed with sonication, the inclusion bodies were extracted with centrifugation. SDS PAGE and Western blot were used to analyze the expression of recombinant protein. The recombinant mhIL 18 inclusion bodies were dissolved with 8 mol/L urea and dialyzed against renaturing buffer. The production of IFN γ by human peripheral blood mononuclear cell (PBMC) was assayed with ELISA after cocultured with conA and renatured protein of different dosages. Results The cloned sequence of mhIL 18 cDNA was identical with the published sequence in GenBank. The recombinant mhIL 18 was expressed in inclusion bodies of E. coli and covered above 40% of total bacterial proteins. Renatured recombinant mhIL 18 could induce the IFN γ production by PBMC. Conclusion mhIL 18 could be expressed as non fusion protein at a high level in E. coli .