Expression and purification of recombinant human IL-18 in pichia pastoris

AIM: To achieve high level expression and purification of recombinant mature human IL-18(mhIL-18)protein in pichia pastoris.METHODS: An Intein Tag was added to the vector.The gene of mhIL-18-Intein was amplified by SOEing and asymmetric PCR.The recombinant expression plasmid pPIC9-IL-18-Intein was constructed by cloning mhIL-18-Intein into pPIC9 vector and was transformed to GS115.The expression of recombinant fusion protein was induced by methanol under an optimum inducing condition.After the protein excreted out of the cells was harvested,SDS-PAGE and Western Blot were used to analyze the expression of recombinant fusion protein.The activity of mhIL-18 purified by affinity chromatography was measured by MTT assays.RESULTS: The cloned sequence of mhIL-18 was identical with the sequence in GenBank.After methanol induction,the fusion protein of mhIL-18 reached the secretion peak of 100 mg/L at 96 h.The purity of the recombinant expressed mhIL-18 was 95% by means of affinity chromatography and the purified mhIL-18 had the biological activity of IL-18.CONCLUSION: We have succeeded in expressing and purifying the recombinant human IL-18 with obvious biological activity in pichia pastoris.