High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system.
Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.
Nagata
Tomoshi
T
Laboratory of Poultry Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan.
Ishikawa
Satoko
S
Shimokawa
Eiko
E
Kamikawa
Makiyo
M
Kikuma
Reiko
R
Muneta
Yoshihiro
Y
Yokomizo
Yuichi
Y
Nakamura
Masayuki
M
Takehara
Kazuaki
K
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D020170:Caspase 1 / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D007371:Interferon-gamma / Q000032:analysis / Q000096:biosynthesis; D020382:Interleukin-18 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera / Q000235:genetics; D015163:Superinfection / Q000662:veterinary
D020382:Interleukin-18; D011994:Recombinant Proteins; D007371:Interferon-gamma; D020170:Caspase 1