Posttranslational processing of recombinant human interferon-gamma in animal expression systems.
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.
James
D C
DC
Research School of Biosciences, University of Kent, Centerbury, United Kingdom. d.c.james@ukc.ac.uk
Goldman
M H
MH
Hoare
M
M
Jenkins
N
N
Oliver
R W
RW
Green
B N
BN
Freedman
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*
D011994:Recombinant Proteins; D007371:Interferon-gamma