A bicistronic expression system for bacterial production of authentic human interleukin-18 Robert B. Kirkpatrick, a, * Patrick J. McDevitt, a Rosalie E. Matico, a Silas Nwagwu, b Stephen H. Trulli, c Joyce Mao, d Dwight D. Moore, e Adam F. Yorke, a Megan M. McLaughlin, f Kristin A. Knecht, f Louis C. Elefante, c Amy S. Calamari, a Jim A. Fornwald, a John J. Trill, a Zdenka L. Jonak, c James Kane, a Pramathesh S. Patel, b Ganesh M. Sathe, d Allan R. Shatzman, c Peter M. Tapley, c and Kyung O. Johanson a a Department of Gene Expression, Protein Biochemistry, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406, USA b Microbial and Cell Culture Development, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA c Microbial Musculoskeletal and Proliferative Diseases, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA d Discovery Genetics, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA e Analytical Methods Development, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA f Department of Comparative Genetics, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA Received 26 June 2002, and in revised form 7 September 2002 Abstract Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co- expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 °C. An alternative bicistronic ex- pression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production. Ó 2002 Elsevier Science (USA). All rights reserved. IL-18 is a potent cytokine that activates both innate and acquired immune responses by stimulating T and natural killer (NK) cells [1,2]. IL-18, formerly known as IFN-c-inducing factor (IGIF), was originally described as a stimulator of the T helper cell type 1 (TH1) re- sponse, characterized by induction of IFN-c production from T and NK cells [3,4]. IL-18, in combination with the structurally unrelated cytokine, IL-12, is a primary mediator of Th1 cytokine production and cell-mediated immune cytotoxicity [5]. IL-18 has also been shown to directly upregulate Fas ligand expression on NK cells [6]. Administration of recombinant IL-18 has anti-tu- mor effects in multiple mouse models, an effect, which appears to involve the induction of Fas-ligand-mediated apoptosis [7–10]. Protein Expression and Purification 27 (2003) 279–292 www.elsevier.com/locate/yprep * Corresponding author. E-mail address: robert_b_kirkpatrick@gsk.com (R.B. Kirkpatrick). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-5928(02)00606-X