PMID- 12597888
OWN - NLM
STAT- MEDLINE
DCOM- 20031009
LR  - 20191106
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 27
IP  - 2
DP  - 2003 Feb
TI  - A bicistronic expression system for bacterial production of authentic human 
      interleukin-18.
PG  - 279-92
AB  - 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.
CI  - Copyright 2002 Elsevier Science (USA)
FAU - Kirkpatrick, Robert B
AU  - Kirkpatrick RB
AD  - Department of Gene Expression, Protein Biochemistry, GlaxoSmithKline 
      Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406, USA. 
      robert_b_kirkpatrick@gsk.com
FAU - McDevitt, Patrick J
AU  - McDevitt PJ
FAU - Matico, Rosalie E
AU  - Matico RE
FAU - Nwagwu, Silas
AU  - Nwagwu S
FAU - Trulli, Stephen H
AU  - Trulli SH
FAU - Mao, Joyce
AU  - Mao J
FAU - Moore, Dwight D
AU  - Moore DD
FAU - Yorke, Adam F
AU  - Yorke AF
FAU - McLaughlin, Megan M
AU  - McLaughlin MM
FAU - Knecht, Kristin A
AU  - Knecht KA
FAU - Elefante, Louis C
AU  - Elefante LC
FAU - Calamari, Amy S
AU  - Calamari AS
FAU - Fornwald, Jim A
AU  - Fornwald JA
FAU - Trill, John J
AU  - Trill JJ
FAU - Jonak, Zdenka L
AU  - Jonak ZL
FAU - Kane, James
AU  - Kane J
FAU - Patel, Pramathesh S
AU  - Patel PS
FAU - Sathe, Ganesh M
AU  - Sathe GM
FAU - Shatzman, Allan R
AU  - Shatzman AR
FAU - Tapley, Peter M
AU  - Tapley PM
FAU - Johanson, Kyung O
AU  - Johanson KO
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Codon)
RN  - 0 (DNA, Complementary)
RN  - 0 (Interleukin-18)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Sulfhydryl Reagents)
RN  - 0 (Ubiquitin)
RN  - 9BZQ3U62JX (Dithionitrobenzoic Acid)
RN  - AE28F7PNPL (Methionine)
RN  - EC 3.4.22.- (CASP4 protein, human)
RN  - EC 3.4.22.- (Caspases)
RN  - EC 3.4.22.- (Caspases, Initiator)
RN  - EC 3.4.22.36 (Caspase 1)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Amino Acid Sequence
MH  - Base Sequence
MH  - Biological Assay
MH  - Caspase 1/metabolism
MH  - Caspases/metabolism
MH  - Caspases, Initiator
MH  - Codon
MH  - Cysteine/metabolism
MH  - DNA, Complementary/metabolism
MH  - Dithionitrobenzoic Acid/pharmacology
MH  - Dose-Response Relationship, Drug
MH  - Enzyme Activation
MH  - Escherichia coli/metabolism
MH  - Gene Library
MH  - Humans
MH  - Interleukin-18/*biosynthesis/*chemistry/metabolism
MH  - Methionine/chemistry
MH  - Molecular Sequence Data
MH  - Plasmids/metabolism
MH  - Protein Biosynthesis
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins/metabolism
MH  - Sulfhydryl Reagents/pharmacology
MH  - Temperature
MH  - Time Factors
MH  - Transcription, Genetic
MH  - Ubiquitin/metabolism
EDAT- 2003/02/25 04:00
MHDA- 2003/10/10 05:00
CRDT- 2003/02/25 04:00
PHST- 2003/02/25 04:00 [pubmed]
PHST- 2003/10/10 05:00 [medline]
PHST- 2003/02/25 04:00 [entrez]
AID - S104659280200606X [pii]
AID - 10.1016/s1046-5928(02)00606-x [doi]
PST - ppublish
SO  - Protein Expr Purif. 2003 Feb;27(2):279-92. doi: 10.1016/s1046-5928(02)00606-x.