Expression of human interferon-α2 in Sf9 cells : characterization of O-linked glycosylation and protein heterogeneities

• Published in: European journal of biochemistry Vol. 217; no. 3; pp. 921 - 927
• Main Authors: SUGIYAMA, K, AHORN, H, MAURER-FOGY, I, VOSS, T
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.11.1993
• Subjects: Antineoplastic drugs. Cytokines; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Production of active biomolecules; Human; Heterologous system; Alpha interferon; Insecta; Cytokine; Spodoptera frugiperda; Glycosylation; Baculovirus; Gene expression; Virus; Baculoviridae; Arthropoda; Lepidoptera; Noctuidae; Recombinant protein; Invertebrata; Vector; Structural analysis
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1993.tb18322.x

Human interferon α2 (IFN‐α2) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte‐derived ‘natural’ IFN‐α2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be O‐glycosylated at the same position as natural human IFN‐α2. Enzymatic cleavage, reverse‐phase HPLC peptide mapping and plasma‐desorption mass spectroscopy analysis revealed the presence of two types of O‐linked carbohydrates. The major O‐linked carbohydrate was found to be the disaccharide galactosyl(β1–3)‐ N ‐acetylgalactosamine, the minor component the monosaccharide N ‐acetylgalactosamine. No evidence for sialylation was found. The non‐glycosylated species representing about 40% of the total material were shown to partially lack the C‐terminal three amino acids. In addition an unglycosylated, reduction‐sensitive dimer was observed. This was formed due to the lack of the N‐terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cys1 and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.