title: Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.
abstract: The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.
authors.affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Electronic address: milenatileva@bio21.bas.bg.
authors.forename: M
authors.initials: M
authors.lastname: Tileva
authors.affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Electronic address: elenakrachmarova@bio21.bas.bg.
authors.forename: E
authors.initials: E
authors.lastname: Krachmarova
authors.affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Electronic address: iivanov@bio21.bas.bg.
authors.forename: I
authors.initials: I
authors.lastname: Ivanov
authors.affiliation: Proteros Biostructures, D-82152 Martinsried, Germany. Electronic address: maskos@proteros.de.
authors.forename: K
authors.initials: K
authors.lastname: Maskos
authors.affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Electronic address: genoveva@bio21.bas.bg.
authors.forename: G
authors.initials: G
authors.lastname: Nacheva
mesh_terms: D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020125:Mutation, Missense; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025842:SUMO-1 Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: C573415:IFNG protein, human; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D007371:Interferon-gamma
keywords: Chaperones; E. coli; Human interferon gamma; Protein aggregation; Recombinant soluble protein; SUMO fusion technology