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. 1996 Feb;5(2):331–340. doi: 10.1002/pro.5560050217

Posttranslational processing of recombinant human interferon-gamma in animal expression systems.

D C James 1, M H Goldman 1, M Hoare 1, N Jenkins 1, R W Oliver 1, B N Green 1, R B Freedman 1
PMCID: PMC2143336  PMID: 8745411

Abstract

We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodopera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.

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