Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous...

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Published in:Protein expression and purification Vol. 211; p. 106339
Main Authors: Chronopoulou, Sofia, Tsochantaridis, Ilias, Tokamani, Maria, Kokkinopliti, Kerasina Despoina, Tsomakidis, Petros, Giannakakis, Antonis, Galanis, Alex, Pappa, Aglaia, Sandaltzopoulos, Raphael
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-11-2023
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Summary:Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form. •Expression of recombinant human IFNα2a in Pichia pastoris.•Purification of secreted recombinant human IFNα2a by affinity chromatography.•Expression and purification of structurally stable and biologically active human IFNα2a as confirmed by DSF analysis and induction of known interferon-stimulated genes (ISGs) in A549 and HT29 cell lines.
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ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2023.106339