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The Study On The Expression And Purification Of Mutant Interleukin-2 With His-tag In Pichia Pastoris

Posted on:2009-07-15Degree:MasterType:Thesis
Country:ChinaCandidate:Q YiFull Text:PDF
GTID:2120360242996905Subject:Biochemistry and Molecular Biology
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Interleukine-2 is an important cytokine of interleukine family,with a molecular mass of~15kDa.IL-2 exerts its effects on many cell types,and the major function of IL-2 is to promote proliferation of T lymphocyte.It can induce the production of TNFα, IFNγ,GM-CSF and other cytokines secreted by T and NK cell.Meanwhile IL-2 can promote proliferation of B cell and enhances the secretion of antibody from B cell. Interleukin-2 is a very important immune regulation molecule.It is crucial for the maintenance of immune homeostasis.Because IL-2 has so many kinds of biological activities,it has promising application in the fields of anti-tumor,infection of pathogenic microbes.In this study,we mainly investigate on human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine;the codon for leucine-18 with methionine;the codon for leucine-19 with serine).We have obtained the following results:The protein MvhIL-2 gene with his-tag was amplified by PCR,and the fusion expression vector pPIC9K-MvhIL-2 was constructed by inserting MvhIL-2 fragment into pPIC9K,then transformed the recombinant plasmid to GS115 by electroporation.After resistence screening in gradient concentration G418,small expression,SDS-PAGE assay,the multi-copy strains were selected for deeper research.Besides the internal character of heteroenous gene sequence itself,expression conditions also play important roles in the expression yield.Different culturable time,pH valves and the dosage of methanol were studied on expression in this paper respectively.We confirmed the best conditions for fermentation as follows:the initial pH of 6.0,the final methanol concentration of 0.5%,72 hours for induction;In this condition,the expressed MvIL-2 was secreted into fermentation broth and reached a yield of above 30%,approximately 32mg/L.In large-scale expression,the interleukin-2 was secreted into fermentation broth by mechanol induction.Expressed IL-2 was isolated and purified by contrifugation,millipore filtration to concentration,Ni2+affinity chromatography.The purity of MvIL-2 reached to 98.6%,and only one band was detected by SDS-PAGE.The western-blotting was used to validate expression of the target protein and the molecular mass of interleukin-2.The result is that we have successfully get the recombinant MvhIL-2.
Keywords/Search Tags:Pichia pastoris, human IL-2 mutant, His tag, Ni2+ affinity chromatography
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