Studies On The Prokaryotic Expression,Purification,Fermentation Technology And Pharmacodynamics Of Recombinant Human Interleukin-18 Binding Protein

Background and Objectives:Interleukin(IL)-18 is a pro-inflammatory cytokine belonging to the IL-1 family(IL-1F),which can effectively stimulate the production of interferon(IFN)-γ.Inflammatory IL-18 exists in the cytoplasm in the inactive form of pro-IL-18 until the activation signal of the inflammasome is induced into mature IL-18,and then these mature forms of IL-18 are secreted out of cells to play their biological functions.IL-18 binds to its receptor on target cells,eventually activating the nuclear factor(NF)-κB signaling pathway,up-regulating the expression of various inflammatory cytokines,and triggering various inflammatory diseases.Interleukin-18 binding protein(IL-18BP)is a natural IL-18 inhibitor with high affinity for IL-18.Among the four isoforms of human IL-18 BP,isoform a has the highest affinity for IL-18,which can neutralize IL-18 and inhibit its induced cytokines,thus playing an anti-inflammatory role.At present,the recombinant IL-18 BP protein is prepared by a eukaryotic expression system,and rarely by a prokaryotic expression system.Because IL-18 BP is a secreted protein composed of eukaryotic cells,it is easy to express into inactive inclusion body structure in the prokaryotic system,and the renaturation efficiency of inclusion body protein is low and the purity is poor,so the safety of medication cannot be guaranteed.Therefore,it is necessary to ensure the biological activity of the recombinant protein and be suitable for large-scale production in the future.In this study,the IL-18 BPa isoform was fused with a small ubiquitin-related modifier protein(SUMO)tag and human Ig G1-Fc fragment,and Escherichia coli(E.coli)was used as host bacteria to establish a pilot-scale fermentation and purification process system,which laid the foundation for obtaining a large number of soluble recombinant protein and realizing large-scale production.Methods:1.Constructing the p ET-20b-Sumo-IL-18BP-Fc expression vector and its transformation into E.coli BL21(DE3)to screen out the strains that stably express recombinant Sumo-IL-18BP-Fc protein;2.Optimizing the induction expression conditions of recombinant Sumo-IL-18BP-Fc protein in a shake flask,including the concentration of inducer IPTG,induction temperature and time;3.Optimizing the fermentation parameters of Sumo-IL-18BP-Fc recombinant protein,including rotation speed,p H value,dissolved oxygen value,induction temperature and time;4.Purifying the recombinant protein by a nickel column affinity chromatography(Ni-NTA)technology to obtain the high-purity recombinant protein IL-18BP-Fc;5.Detecting the ability of the recombinant protein IL-18BP-Fc to inhibit the production of IFN-γ by human acute myeloid leukemia cell(KG-1a)after binding to IL-18 in vitro by ELISA;6.Analyzing the pharmacokinetics of recombinant IL-18BP-Fc protein in vivo and determining its half-life in vivo;7.Establishing a mouse model of inflammatory bowel disease,and detecting the biological activity of IL-18BP-Fc recombinant protein in vivo by intraperitoneal and hypodermic injection.Results:1.The recombinant strain of p ET-20b-Sumo-IL-18BP-Fc was successfully constructed,and the expressed recombinant protein of Sumo-IL-18BP-Fc was detected by SDS-PAGE.2.The optimal expression conditions of recombinant Sumo-IL-18BP-Fc protein induced in a shake flask were 0.5 m M IPTG at 30℃ for 5 h;3.The optimum fermentation conditions of recombinant Sumo-IL-18BP-Fc protein were 0.5 m M IPTG at 20 ℃ for 26 h.After fermentation,the soluble expression of the recombinant protein accounted for more than 85 % of the total protein,and the expression amount was about 1 g per liter of bacterial liquid;4.The recombinant IL-18BP-Fc protein was purified by Ni-NTA affinity chromatography with a purity of more than 90 %,and the sequence was confirmed to be correct by mass spectrometry;5.The recombinant protein IL-18BP-Fc had good stability and biological activity in vitro;6.The half-life of the recombinant protein IL-18BP-Fc in vivo was about 26 h;7.The recombinant protein IL-18BP-Fc can effectively treat inflammatory bowel disease in mice.Conclusions:In this study,the recombinant strain of Sumo-IL-18BP-Fc was constructed,and the induced expression conditions of the recombinant protein were optimized.A large number of soluble recombinant protein Sumo-IL-18BP-Fc was obtained in the host of E.coli,and a pilot fermentation process system of Sumo-IL-18BP-Fc was established to obtain high-yield recombinant protein Sumo-IL-18BP-Fc.Through Ni-NTA affinity chromatography,high-purity recombinant IL-18BP-Fc protein was obtained,which can specifically bind IL-18 and inhibit its activity,and had good biological activity in vivo and in vitro.