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Isolation and Characterization of an Insect Cell Line Able to Perform Complex N-Linked Glycosylation on Recombinant Proteins

Bio/Technology volume 14, pages 197–202 (1996)Cite this article

Abstract

Site specific characterization of the N-glycan structures in human interferon γ (IFN-γ) derived from baculovirus-infected insect cells was performed using a combination of reverse-phase, high-performance liquid chromatography (rHPLC) and matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry. IFN-γ was produced in two cell lines, an Estigmena acmz-derived subclone (Ea4), and Spodopterafrugiperda cells (Sf9). Both IFN-γ N-glycosylation sites (Asn25and Asn97) were characterized. Site-specific differences were observed in both the percentage of sites occupied by N-linked glycans and the types of structure associated with each site. The glycosylation capabilities and glycan processing of Sf9 were limited to the generation of chitobiose [GlcNAc2], truncated tri-mannose core [Man3GlcNAc2], or oligomannose structures. The glycosylation abilities of Ea4 cells were more extensive, producing IFN-γ molecules incorporating oligosaccharides with GlcNAc and Gal residues on the outer arms (hybrid or complex type N-glycans), as well as oligomannose N-glycans. Incorporation of an αl–6 linked fucose residue (<70% in Sf9 and <88% in Ea4) was confined to the Asn25 glycosylation site. These findings demonstrate the more extensive N-glycosylation capabilities of the E1 acrea–derived Ea4, compared to current insect cell lines used for the expression of recombinant proteins.

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Author notes

  1. Nigel Jenkins

    Present address: Department of Biological Sciences, de Montfort University, The Gateway, Leicester, LEI 9BH, U.K.

  2. Barrie C. Rooney: (e-mail: B.C.Rooney@ukc.ac.uk).

Authors and Affiliations

  1. Research School of Biosciences, Biological Laboratory, The University of Kent, Canterbury, CT2 7NJ, U.K.

    Olotu W. Ogonah, Robert B. Freedman & Kirit Patel

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Ogonah, O., Freedman, R., Jenkins, N. et al. Isolation and Characterization of an Insect Cell Line Able to Perform Complex N-Linked Glycosylation on Recombinant Proteins. Nat Biotechnol 14, 197–202 (1996). https://doi.org/10.1038/nbt0296-197

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