Expression,Purification,and Biological Activity Of Recombinant Human Interleukin 1β In Pichia Pastoris

Interleukin-1beta(IL-1β) is one kind of important promoting pro-inflammatory cytokines, which participate in a variety of physiological and pathological response. Currently, the human IL-1β is mainly obtained from prokaryotic expression system by Escherichia coli which have been used extensively as the cellular host for foreign protein expression. The dominant advantages of prokaryotic expression based on E. coli are rapid growth rate, capacity for continuous fermentation and relatively low cost. However, E. coli expression system has the low levels of productivity, containing endotoxin and difficult of purification. So, in order to obtain higher yield recombinant human interleukin-1beta(rhIL-1β), we designed and synthesized rhIL-1β based on the amino acid sequence of human mature IL-1β, and used Pichia pastoris to express. Firstly, we exported hIL-1β mRNA to pPIC9、pPIC9K and pPICZαA vectors,and built rhIL-1β recombinant plasmids. Secondly, the recombinant plasmids were transformed into Pichia pastoris GS115, SMD1168 and X-33 strain via electroporation. From recombinant yeast screened correct and efficient expression of engineering strain, then we discussed the appropriate conditions of 3L fermentation, purification process of product and biological function in vitro.In this study, the recombinant expression vectors pPIC9-hIL-1β, pPIC9K-hIL-1β and pPICZαA-hIL-1β were identified and integrated into pichia GS115、SMD1168 and X-33 genome through electrotransformation. In order to get the recombinant strains expressing rhIL-1β, a series of processes were performed: identification and filtering genome with PCR, the MD flat filtering monoclone, induction and training with BMGY medium, proteins identification by SDS-PAGE and western blot. Finally, the highest level expression of pPICZαA/hIL-1β in X-33 P. pastoris was obtained.To study best expression levels of rhIL-1β in the 3L fermenter, five conditions in the induction phase were studied, such as different starting cell density, temperature, pH, dissolved oxygen volume fraction(DO) and induction concentrations of methanol. The fermentation process was optimized to increase product yield. With the conditions of initial cell density of 600, at 26℃, pH 5.0, dissolved oxygen of 20%, methanol concentration of 0.25%, the expression level of rhIL-1β could reach up to 280mg/L after 72 h induction.The conditions of separating and purifying rhIL-1β were preliminarily researched and the results showed that after centrifugation hypothermia, phosphate / ethanol formation the aqueous two-phase, 10 kD membrane ultrafiltration and concentration, and DEAE weak anion exchange chromatography, the purity of rhIL-1β could be 95% and the protein yield was up to 75%.Finally, the biological activity of the synthesized rhIL-1β was conducted by MTT assay and Hoechst33258 staining. MTT assay showed that rhIL-1β could inhibit the proliferation of human hepatoma cell line HepG2 and mouse melanoma cell line B16. The results of microscopic observation of rhIL-1β-treated HepG2 and B16 cells revealed that rhIL-1β could promote apoptosis in both two cancer cell lines.But above all, this study set a basis for industrial production and biological function research of rhIL-1β and provide experimental basis for new antineoplastic drugs development.