Production of a biologically active human interleukin 18 requires its prior synthesis as PRO-IL-18.
Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th(1)cytokines, sharing structural features with the IL-1 family of proteins. Unlike most other cytokines, IL-18 and IL-1beta lack a signal peptide, have an all beta-pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1beta). These precursors are cleaved by caspase-1 (IL-1beta-converting enzyme, ICE) to form the biologically active mature cytokines. Direct expression of mature recombinant human IL-18 in E. coli resulted in a partially active cytokine. We tested the possibility that correct folding of huIL-18 requires its prior synthesis as pro-IL-18. Because caspase-1 is not readily available, we constructed an expression vector encoding human pro-IL-18 in which the caspase-1 cleavage site was mutated into a factor Xa site. To facilitate purification, the mutated pro-IL-18 cDNA was fused in frame to a glutathione-S-transferase (GST) coding sequence. The GST-pro-IL-18 fusion protein was expressed in E. coli, captured on glutathione agarose and mature human IL-18, exhibiting high biological activity was released upon cleavage with factor Xa. This result indicates that correct folding of huIL-18 occurs at the level of pro-IL-18 and provides a practical way to produce biologically active huIL-18.
Liu
B
B
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel.
Novick
D
D
Kim
S H
SH
Rubinstein
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D020170:Caspase 1 / Q000737:chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D020382:Interleukin-18 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D018076:DNA, Complementary; D020382:Interleukin-18; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D005982:Glutathione Transferase; D015951:Factor Xa; D020170:Caspase 1; D005978:Glutathione