A bicistronic expression system for bacterial production of authentic human interleukin-18

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 I 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 coexpression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degreesC. An alternative bicistronic expression 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. (C) 2002 Elsevier Science (USA). All rights reserved.