title: Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli.
abstract: Type I (alpha/beta) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-alpha (SasaIFN-alpha1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-alpha1 (rSasaIFN-alpha1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-alpha subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-alpha, the mature form of the SasaIFN-alpha2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-alpha2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-alpha2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 x 10(6) U mg(-1) against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-alpha2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-alpha2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.
authors.affiliation: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Ei Lin
authors.initials: EL
authors.lastname: Ooi
authors.forename: Noel
authors.initials: N
authors.lastname: Verjan
authors.forename: Ikuo
authors.initials: I
authors.lastname: Hirono
authors.forename: Tomonori
authors.initials: T
authors.lastname: Nochi
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Kondo
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyono
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Yuki
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics*; D029941:Fish Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D018061:Infectious pancreatic necrosis virus / Q000187:drug effects; D007370:Interferon Type I / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D016898:Interferon-alpha / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D064547:Myxovirus Resistance Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D020317:Salmo salar / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D029941:Fish Proteins; D007370:Interferon Type I; D016898:Interferon-alpha; D064547:Myxovirus Resistance Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins