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- Title
Expression, purification and characterization of human interferon-γ in Pichia pastoris.
- Authors
DAN WANG; HUI REN; JING-WEI XU; PENG-DA SUN; XUE-DONG FANG
- 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 1x107-1.4x107 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.
- Subjects
PICHIA pastoris; INTERFERONS; IMMUNOMODULATORS; ANTIVIRAL agents; ANTINEOPLASTIC agents; ALCOHOL oxidase
- Publication
Molecular Medicine Reports, 2014, Vol 9, Issue 2, p715
- ISSN
1791-2997
- Publication type
Article
- DOI
10.3892/mmr.2013.1812