Pruification, Refolding And Bioactivity Assay Of Recombinant Human Interleukin 18

Objective Interleukin-18 is a novel cytokine, which was previously known as interferon-γ-inducing factor ( IGIF ) because of its potent inducing interferon-γproduction. It has been revealed that human IL-18 possesses multiple potent biological activities such as augmenting IFN-γ and GM-CSF production in peripheral blood mononuclear cells and enriched T-cells ,and inhancing NK cell cytotoxicity. Its biologic activities are similar to IL-12. IGIF shares common structure of IL-1 family of proteins. IGIF is synthesized as a biologically inactive precursor molecule which requires cleavage at an Asp-X site by interleukin-1β-converting enzyme(ICE). Recent investigations have indicated that IL-18 plays important roles in antitumer, antimicrobiol and immune regulation. These results indicated that rhIL-18 can be used to cure clinical diseases. In order to produce rhIL-18 in vitro, it is important to set up a productive method producing recombinant human interleukin-18 with high efficiency and low cost on a large scale . At present study, we have set up a good renature and purification system and bioactivity assay method. The established method for purifying rhIL-18 is simple and effective and easy to scale up.Methods and results1. Establishment of IL-18 expression system and scanning of high effective IL-18-expressing engineering bacteriaThe cDNA of IL-18 encoding 157 amino acids of mature IL-18 was synthesized by chemical method according to the sequence of IL-18 gene. Synthesized cDNA was inserted into expressive vector pBV220 and then the recombinant vector was transformed into E.coli BL21. IL-18 was highly and stably expressed in BL21 and was about 30% of the total somatic proteins.2. The fermentation of engineering bacteria and induction of rhIL-18 expressionThe rhIL-18 expression could be induced by raising the temperature to 42 °C for the recombinant plasmid composed of a temperature-sensitive PlPr promoter. The engineering bacteria was first incubated in LA culture in shake flask at 30°C for 10 hours.then transfered it to new LA culture according to 1% to amplify to optical density as the seed batch. The bacteria were transfered to LB+M9 medium in the fermentor and amplified at 30 °C by controlling Dissolved O2 and pH to keep the bacteria grow fast.When the cell density was optical ,the temperature was raised to 42 °C inducing rhIL-18 expression.The inducing time was 4 hours. Conditions of high cell density and high expression were established. We can obtained 10 g wet cell weihgt per liter.3. The purification and renaturation of rhIL-18The expressed rhIL-18 products existed in the form of inclusion bodies. For purification of inclusion bodies, the harvest cells were disrupted using lysozyme and sonication, and the rhIL-18 inclusion bodies were extracted by centrifugation and washed with 1% TritonX-100 and solubilized in 8mol/L urea. The purity of rhIL-18 in extracted inclusion bodies was above 70%. The solubilized IL-18 was first purified by gel filtration chromatography under the denaturing conditions and renaturalized by dialysis and dilution. The renaturalized rhIL-18 was purified by Ion exchange chromatography further. After above purification, the purity of rhIL-18 was analyzed by SDS-PAGE and HPLC size exclusion chromatography. The results demonstrated the purity of rhIL-18 was above 95%.4. The detection of three lots of rhIL-18 products according to theNational Standard of Biological ProductsTo confirm whether rhIL-18 produced by our process could reach to the National Standard of Biological Products. Three lots of rhIL-18 were produced using our process and their purification was detected. The results showed purity of all rhIL-18 from three lots was above 95%. The residual host somatic protein of the product was less than 0.1%. Residual somatic DNA was less than lOng/mg. Endotoxin was less than lOEU/mg. Purity of rhIL-18 products have reached to the National Standard of Biological Products. It demonstrated that our process for rhIL-18 production and purification is stable.5. Evaluation of rhIL-18 biological activityThe biological activity of the purified product can be assayed by its activity of argumenting PBMC proliferation and inducing IFN-y production.The assay of detecting the IFN-ylevel secreted by PBMC is sensitive.PBMCs were made by normal method and adjusted cell concentration to 3*106 /ml.The cells were incubated in RPMI1640 medium added lOU/ml IL-2 and different concentration of rhIL-18.The IFN-y level of the culture supernatant was assayed by ELISA. The rhIL-18 could increase significantly IFN-y level secreted by PBMCs in a dose-dependent manner in the presense of lOU/ml IL-2.Conclusion and significance1. IL-18 expression system in E.coli has successfully established and high effective IL-18-expressing engineering bacteria have scanned.2.We have set up a good renature and purification process after comparing various method. The process (including extracting inclusion bodies, gel filtration, refolding, ion-exchange chromatography) is simple, effective and easy to scale up.3.It is found that the bioassay of rhIL-18 can be performed by detecting IFN-ylevel secreted with PBMC under the induction of it.4. Three lots of rhIL-18 were produced using our process and the purities of all rhIL-18 have reached to the National Standard of Biological Products. Itdemonstrated that our process for rhIL-18 production is stable.5.This study has laid a good foundation of bulk production and certificate of new drugs of rhIL-18.