Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli.
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.
Ooi
Ei Lin
EL
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.
Verjan
Noel
N
Hirono
Ikuo
I
Nochi
Tomonori
T
Kondo
Hidehiro
H
Aoki
Takashi
T
Kiyono
Hiroshi
H
Yuki
Yoshikazu
Y
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*
D000998:Antiviral Agents; D029941:Fish Proteins; D007370:Interferon Type I; D016898:Interferon-alpha; D064547:Myxovirus Resistance Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins