title: Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris.
abstract: BACKGROUND
  Interferon (IFN)-α has been commonly used as an antiviral drug worldwide; however, its short half-life in circulation due to its low molecular weight and sensitivity to proteases impacts its efficacy and patient compliance.
  
  
  RESULTS
  In this study, we present an IgG1 Fc fusion strategy to improve the circulation half-life of IFN-α. Three different forms of IgG1 Fc fragments, including the wild type, aglycosylated homodimer and aglycosylated single chain, were each fused with IFN-α and designated as IFN-α/Fc-WT, IFN-α/Fc-MD, and IFN-α/Fc-SC, respectively. The recombinant proteins were expressed in Pichia pastoris and tested using antiviral and pharmacokinetic assays in comparison with the commercial pegylated-IFN-α (PEG-IFN-α). The in vitro study demonstrated that IFN-α/Fc-SC has the highest antiviral activity, while IFN-α/Fc-WT and IFN-α/Fc-MD exhibited antiviral activities comparable to that of PEG-IFN-α. The in vivo pharmacokinetic assay showed that both IFN-α/Fc-WT and IFN-α/Fc-MD have a longer half-life than PEG-IFN-α in SD rats, but IFN-α/Fc-SC has the shortest half-life among them. Importantly, the circulating half-life of 68.3 h for IFN-α/Fc-MD was significantly longer than those of 38.2 h for IFN-α/Fc-WT and 22.2 h for PEG-IFN-α.
  
  
  CONCLUSIONS
  The results demonstrate that the elimination of N-glycosylation by mutation of putative N-glycosylation site further prolongs the half-life of the IFN-α/Fc fusion protein and could present an alternative strategy for extending the half-life of low-molecular-weight proteins expressed by P. pastoris for in vivo studies as well as for future clinical applications.
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Jia
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China. gmp@ustc.edu.cn.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China. gmp@ustc.edu.cn.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China. gmp@ustc.edu.cn.
authors.forename: Yugang
authors.initials: Y
authors.lastname: Guo
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Department of Pharmacy, Anhui Medical College, Hefei, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Song
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China.
authors.forename: Changsheng
authors.initials: C
authors.lastname: Shao
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Ma
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.
authors.forename: Mingyang
authors.initials: M
authors.lastname: Shi
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Miao
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Tian
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China. xiaow@ustc.edu.cn.|Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China. xiaow@ustc.edu.cn.|Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China. xiaow@ustc.edu.cn.
authors.forename: Weihua
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000818:Animals; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics*; D009154:Mutation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics*
chemical_list: D007141:Immunoglobulin Fc Fragments; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins
keywords: Circulation half-life; Fusion protein; Glycosylation; IFN-α/Fc; Pichia pastoris