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Codon Optimization, Expression, Purification And Characterization Of Recombinant Human IL-25in Pichia Pastoris

Posted on:2012-03-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y S LiuFull Text:PDF
GTID:1220330434471416Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Abstract:IL-25, formerly known as IL-17E, shares the sequence similarity with IL-17. It belongs to the IL-17cytokine family, has only15-20%homology to other five members, indicating it has special function. Indeed, the biological activity of IL-25are markedly different from IL-17A and other IL-17family cytokines.IL-25is able to induce type-2immunity and plays a decisive role both in the induction of a protective response against parasites and in the development of allergic diseases. IL-25can induce NF-κB activation and stimulate the production of IL-8and G-CSF. Further research found that IL-25induces the production of other type-2cytokines, including IL-4, IL-5and IL-13in multiple tissues, which stimulate the expansion of eosinophils, splenomegaly and gut inflammation. IL-25is not only necessary for the induction of type-2immunity, which is required for the efficient clearance of parasitic worm infections, but also required for the generation of an innate type cell which may contribute to the initial source of type-2cytokine at the initiation of an adaptive type-2-mediated response. This highlights IL-25as a potential target in modulating pulmonary inflammation and airways hyperreactivity during allergic asthma.IL-25plays central roles in the regulation of type2and type9T helper cell responses, but the detailed pathways by which I1-25positively regulates Th2responses remain poorly described. IL-25acts as a negative regulator of inflammatory cell responses. It can promotes the immune responses that cause asthma and allergy, but at the same time it can limit the type of inflammation associated with debilitating human diseases such as inflammatory bowel disease, diabetes and MS(multiple sclerosis). IL-25can be helpful or damaging to the host, which depending on how and where it interacts with other components of the immune response. More investigation in different settings are required for further confirmation of this dual role of IL-25. This could impacts the future design of therapeutic strategies targeting I1-25/IL-25R pathway.In this study, we try to express recombinant IL-25using the methylotropic yeast Pichia pastoris expression system. Three Pichia pastoris strains, GS115, KM71and X-33were transformed with two vectors of pPIC9K or pPICZaA, both of which are under the control of AOX1(alcohol oxidase), inserted by the cDNA fragment coding for mature IL-25. After screening on antibiotics, the transformants was cultured in BMGY and induced by methanol in BMMY. Soluble rhIL-25was expressed as a secreted protein as both monomer and dimmer form by the strain of X-33transformed by pPICZaA, but the expression level was so low that the rIL-25can only be detected by silver-staining and confirmed by western-blot analysis.By analysis of the lysate of transformants, we found that even the expression level of rhIL-25intra-cellular is also low, which indicates the coden bias of P. pastoris limited higher expression of target protein. So, we optimized the coden of IL-25for P. pastoris coden preference. Finally we got high expression strain of X-33transformed by pPICZaA. Soluable rhIL-25was secreted in the supernant of culture.To get enough rhIL-25, we performed high density fermentation in A5L fermenter with a initial volume of2L medium. The rhIL-25can be detected in the culture supernatant16h after induction and the induction period was lasting for40h. The supernatant was separated by centrifugation and desalt by a G-25gel filter. Then the buffer changed protein was flow through Q Sepharose Fast Flow which was balanced with50mM Tris-Cl,1mM EDTA, pH8.8. The contaminant was binded to the ion-exchange medium while rhIL-25remaining in the flow through alone. After dehydration by freeze dry, the purified recombinant protein was run a Sephacryal S-100size-exclusion chromatography to desalt and polish. Recombinant IL-25was then aliquoted and lyophilized with mannitol as vehicle.The biological activity of rhIL-25was measured in vitro using human embryo lung fibroblast HFL-I and293cell. The mRNA expression level of G-CSF was examined by a quantitative real-time PCR and ELISA, both results showed rhIL-25can induce G-CSF transcription and expression, which was in agreement with reported research. We also characterize IL-25by its special binding activity with its receptor IL-17RB in a pull-down assay.We accidentally found that rhIL-25can induce apoptosis in two breast cancer cells, MD231and MCF7. Western blot results showed IL-25R expression is higher in this two cells compared with other cells. Caspases8and3was activated during apoptosis, which indicating the potential signal transduction pathway.In summary, we have archived a quick and economic way to largely produce rhIL-25with biological activity, which may facilitate further studies of its structure-function relationship, and that of clinic applications.
Keywords/Search Tags:IL-25, Pichia pastoris, recombinant expression, fermentation, protein purification, coden optimization, bioactivity assay, apoptosis
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