Posttranslational processing of recombinant human interferon-gamma in animal expression systems.
Protein Sci
; 5(2): 331-40, 1996 Feb.
Article
in En
| MEDLINE
| ID: mdl-8745411
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein Processing, Post-Translational
/
Interferon-gamma
/
Cloning, Molecular
/
Spodoptera
/
Mammary Glands, Animal
Limits:
Animals
/
Female
/
Humans
Language:
En
Journal:
Protein Sci
Journal subject:
BIOQUIMICA
Year:
1996
Type:
Article
Affiliation country:
United kingdom