title: A bicistronic expression system for bacterial production of authentic human interleukin-18.
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 degrees C. 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.
authors.affiliation: Department of Gene Expression, Protein Biochemistry, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406, USA. robert_b_kirkpatrick@gsk.com
authors.forename: Robert B
authors.initials: RB
authors.lastname: Kirkpatrick
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: McDevitt
authors.forename: Rosalie E
authors.initials: RE
authors.lastname: Matico
authors.forename: Silas
authors.initials: S
authors.lastname: Nwagwu
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Trulli
authors.forename: Joyce
authors.initials: J
authors.lastname: Mao
authors.forename: Dwight D
authors.initials: DD
authors.lastname: Moore
authors.forename: Adam F
authors.initials: AF
authors.lastname: Yorke
authors.forename: Megan M
authors.initials: MM
authors.lastname: McLaughlin
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Knecht
authors.forename: Louis C
authors.initials: LC
authors.lastname: Elefante
authors.forename: Amy S
authors.initials: AS
authors.lastname: Calamari
authors.forename: Jim A
authors.initials: JA
authors.lastname: Fornwald
authors.forename: John J
authors.initials: JJ
authors.lastname: Trill
authors.forename: Zdenka L
authors.initials: ZL
authors.lastname: Jonak
authors.forename: James
authors.initials: J
authors.lastname: Kane
authors.forename: Pramathesh S
authors.initials: PS
authors.lastname: Patel
authors.forename: Ganesh M
authors.initials: GM
authors.lastname: Sathe
authors.forename: Allan R
authors.initials: AR
authors.lastname: Shatzman
authors.forename: Peter M
authors.initials: PM
authors.lastname: Tapley
authors.forename: Kyung O
authors.initials: KO
authors.lastname: Johanson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D020170:Caspase 1 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D053471:Caspases, Initiator; D003062:Codon; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D020382:Interleukin-18 / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D003062:Codon; D018076:DNA, Complementary; D020382:Interleukin-18; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D025801:Ubiquitin; D004228:Dithionitrobenzoic Acid; D008715:Methionine; C093516:CASP4 protein, human; D020169:Caspases; D053471:Caspases, Initiator; D020170:Caspase 1; D003545:Cysteine