High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system |
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Authors: | Nagata Tomoshi Ishikawa Satoko Shimokawa Eiko Kamikawa Makiyo Kikuma Reiko Muneta Yoshihiro Yokomizo Yuichi Nakamura Masayuki Takehara Kazuaki |
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Institution: | Laboratory of Poultry Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. |
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Abstract: | 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. |
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