title: Expression, purification and characterization of human interferon-γ in Pichia pastoris.
abstract: Human interferon-γ (hIFN-γ) is a multifunctional protein known to possess immunoregulatory, antiviral and anticancer functions. In the present study, in order to explore the biological roles of hIFN-γ and its mechanisms of action, IFN-γ was cloned and expressed in Pichia pastoris (P. pastoris) under the control of alcohol oxidase promoter 1 (AOX1). The protein was secreted by two different signal peptides, the native secretion signal peptide of hIFN-γ and the Saccharomyces cerevisiae α signal peptide. Following 96 h of methanol induction, Tricine-SDS-PAGE Coomassie staining, western blot analysis and N-terminal protein sequencing revealed that the level of recombinant hIFN-γ (rhIFN-γ) secreted by the native secretion signal was barely detectable, while the α signal peptide secreted ~300 mg/l. rhIFN-γ was purified by Vivaflow 200, SP Sepharose Fast Flow and Vivaspin 2 ml, yielding >96% of a highly purified rhIFN-γ preparation, with a specific activity of 1 x 10(7)-1.4 x 10(7) IU/mg protein as determined by an antiviral assay. The results demonstrated that the experimental procedures developed are capable of producing a large quantity of active rhIFN-γ from P. pastoris.
authors.affiliation: Department of General Surgery, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of General Surgery, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ren
authors.affiliation: Department of General Surgery, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Jing-Wei
authors.initials: JW
authors.lastname: Xu
authors.affiliation: Department of General Surgery, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Peng-Da
authors.initials: PD
authors.lastname: Sun
authors.affiliation: Department of General Surgery, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Xue-Dong
authors.initials: XD
authors.lastname: Fang
mesh_terms: D000595:Amino Acid Sequence; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: C573415:IFNG protein, human; D011994:Recombinant Proteins; D007371:Interferon-gamma