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
  • View Affiliations

  • Published online on: November 19, 2013     https://doi.org/10.3892/mmr.2013.1812
  • Pages: 715-719
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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.
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2014-February
Volume 9 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Spandidos Publications style
Wang D, Ren H, Xu J, Sun P and Fang X: Expression, purification and characterization of human interferon-γ in Pichia pastoris. Mol Med Rep 9: 715-719, 2014
APA
Wang, D., Ren, H., Xu, J., Sun, P., & Fang, X. (2014). Expression, purification and characterization of human interferon-γ in Pichia pastoris. Molecular Medicine Reports, 9, 715-719. https://doi.org/10.3892/mmr.2013.1812
MLA
Wang, D., Ren, H., Xu, J., Sun, P., Fang, X."Expression, purification and characterization of human interferon-γ in Pichia pastoris". Molecular Medicine Reports 9.2 (2014): 715-719.
Chicago
Wang, D., Ren, H., Xu, J., Sun, P., Fang, X."Expression, purification and characterization of human interferon-γ in Pichia pastoris". Molecular Medicine Reports 9, no. 2 (2014): 715-719. https://doi.org/10.3892/mmr.2013.1812