title: Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli.
abstract: Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an Escherichia coli expression system. Here we describe the expression of soluble and active recombinant IFN-con in E. coli. The IFN-con gene sequence was optimised for expression in E. coli, which was then cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein and under strong T7lac promoter. The SUMO-IFN-con fusion protein was efficiently expressed using the SHuffle™ E. coli strain and existed in soluble form as 86-88% of the total IFN-con. After removal of the SUMO fusion partner, approximately 50mg of recombinant IFN-con of at least 98% purity (by RP-HPLC) was obtained from a 1L fermentation culture. Using an A549/EMCV antiviral assay, the specific activity of the recombinant IFN-con was determined to be 960×10(6) IU/mg as calculated to NIBSC standard for IFN-con (3×10(5)pfu/mL virus titre). Comparison of the antiviral activity of the produced IFN-con to IFN α-2a showed that IFN-con displays 2.8 times greater activity, which is in good agreement with what has been reported in the literature for pure protein. IFN-con expression in a soluble form from E. coli allowed us to use a simple, two-step purification process to yield highly pure and active IFN-con which is more efficient than obtaining IFN-con from inclusion bodies.
authors.affiliation: UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK; PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK.
authors.forename: Karolina
authors.initials: K
authors.lastname: Peciak
authors.affiliation: PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK.
authors.forename: Rita
authors.initials: R
authors.lastname: Tommasi
authors.affiliation: PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK.
authors.forename: Ji-won
authors.initials: JW
authors.lastname: Choi
authors.affiliation: UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK; PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK.
authors.forename: Steve
authors.initials: S
authors.lastname: Brocchini
authors.affiliation: PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK. Electronic address: emmanuelle.laurine@polytherics.com.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Laurine
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics
chemical_list: D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C050739:interferon alfacon-1
keywords: Consensus interferon; E. coli; Expression; SUMO; Soluble