James, D.C. and Goldman, M.H. and Hoare, M. and Jenkins, N. and Oliver, R.W.A. and Green, B.N. and Freedman, R.B. (1996) Posttranslational processing of recombinant human interferon-gamma in animal expression systems. Protein Science, 5 (2). pp. 331-340. ISSN 0961-8368.
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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 Spodoptera 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.
| Item Type: | Article |
|---|---|
| Subjects: | Q Science > QP Physiology (Living systems) > QP517 Biochemistry |
| Divisions: | Faculties > Science Technology and Medical Studies > School of Biosciences |
| Depositing User: | R.F. Xu |
| Date Deposited: | 04 Jun 2009 16:10 |
| Last Modified: | 04 Jun 2009 16:10 |
| Resource URI: | http://kar.kent.ac.uk/id/eprint/19260 (The current URI for this page, for reference purposes) |
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