Expression and purification of recombinant human ILª²18 in pichia pastoris

    PENG Yan, WANG Yong, SONG Fangª²Zhou, WANG Yaª²Ping

    Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing University of Medical Sciences, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To achieve high level expression and purification of recombinant mature human ILª²18(mhILª²18)protein in pichia pastoris. METHODS: An Intein Tag was added to the vector. The gene of mhILª²18ª²Intein was amplified by SOEing and asymmetric PCR. The recombinant expression plasmid pPIC9ª²ILª²18ª²Intein was constructed by cloning mhILª²18ª²Intein 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, SDSª²PAGE and Western Blot were used to analyze the expression of recombinant fusion protein. The activity of mhILª²18 purified by affinity chromatography was measured by MTT assays. RESULTS: The cloned sequence of mhILª²18 was identical with the sequence in GenBank. After methanol induction, the fusion protein of mhILª²18 reached the secretion peak of 100 mg/L at 96 h. The purity of the recombinant expressed mhILª²18 was 95% by means of affinity chromatography and the purified mhILª²18 had the biological activity of ILª²18. CONCLUSION: We have succeeded in expressing and purifying the recombinant human ILª²18 with obvious biological activity in pichia pastoris. ......