title: Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells.
abstract: Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ.
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia. Electronic address: sebastian.stifter@monash.edu.
authors.forename: Sebastian A
authors.initials: SA
authors.lastname: Stifter
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia. Electronic address: jodee.gould@monash.edu.
authors.forename: Jodee A
authors.initials: JA
authors.lastname: Gould
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia. Electronic address: niamh.mangan@monash.edu.
authors.forename: Niamh E
authors.initials: NE
authors.lastname: Mangan
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia. Electronic address: hugh.reid@monash.edu.
authors.forename: Hugh H
authors.initials: HH
authors.lastname: Reid
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia; Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff CF14 4XN, UK. Electronic address: jamie.rossjohn@monash.edu.
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia. Electronic address: paul.hertzog@monash.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hertzog
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia. Electronic address: nicole.deweerd@monash.edu.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: de Weerd
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D006639:Histidine / Q000235:genetics; D007313:Insecta / Q000166:cytology / Q000235:genetics; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D016899:Interferon-beta
keywords: Biologically active; Endotoxin-free; Insect cell expression; Interferon beta; Natively folded; Soluble protein