Expression, purification and characterization of human interferon-γ in Pichia pastoris

DAN WANG, HUI REN, JING-WEI XU, PENG-DA SUN, XUE-DONG FANG
2013 Molecular Medicine Reports  
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
more » ... revisiae 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 1x10 7 -1.4x10 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.
doi:10.3892/mmr.2013.1812 pmid:24253448 fatcat:3ok746mbgjbjpe4zu6mwp566py