Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice

Abstract

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Keywords: Human IgG1 Fc fragment; IL-18BP; SUMO tag; Ulcerative colitis.