Posttranslational processing of recombinant human interferon‐γ in animal expression systems

Publisher Website
Google Scholar
Abstract
We have characterized the heterogeneity of recombinant human interferon‐γ (IFN‐γ) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus‐infected Spodoptera frugiperda (Sf9) insect cells. Analyses of whole IFN‐γ proteins by electrospray ionization‐mass spectrometry (ESI‐MS) from each recombinant source revealed heterogeneous populations of IFN‐γ 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‐γ proteins; MALDI‐MS analyses of released, desialylated N‐glycans and of deglycosylated IFN‐γ 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‐γ in the mammalian expression systems yielded polypeptides C‐terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN‐γ molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high‐mannose type N‐glycans. In contrast, IFN‐γ 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.