Abstract
We have shown in previous studies that recombinant human interferon-γ (IFN-γ) expressed in CHO cells displays macroheterogeneity caused by variable N-glycosylation (Curling et al., 1990). In this paper, FAB-Mass Spectrometry of IFN-γ glycopeptides was used to assign the N-glycosylation preference of individual asparagine (Asn) sites within the IFN-γmolecule. Whilst both Asn28 and Asn100 N-glycosylation sites were used in doubly-glycosylated IFN-γ, a clear preference for the Asn28 site was apparent in the singly-glycosylated IFN-γ glycoform. The extent of glycosylation at the Asn28 site remained relatively stable during batch culture, whereas the percentage of Asn100 sites glycosylated decreased from 60% to 23% over the same period. These results demonstrate that marked differences exist in the capacity of CHO cells to glycosylate at individual Asn sites during cell culture.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Bause, E. (1984) Model studies on N-glycosylation of proteins. Biochem. Soc. Trans. 12, 514–517.
Curling, E.M.A., Hayter, P.M., Strange, P.G., Baines, A.J., Bull, A.T. and Jenkins, N. (1990) Recombinant interferon-γ: glycosylation and proteolytic processing leads to heterogeneity in batch culture. Biochem. J. 272, 333–337.
Ealick, S.E., Cook, W.J., Vijaykumar, S., Carson, M., Nagabhushan, T.L., Trotta, P.P. and Bugg, C.E. (1991) 3-Dimensional structure of recombinant human interferon-gamma. Science 252,698–702.
Gavel, Y. and von Heijne, G. (1990) Sequence differences between glycosylated and nonglycosylated Asn-x-Thr/Ser acceptor sites - implications for protein engineering. Protein Engineering 3, 433–442.
Goochee, C.F. and Monica, T. (1990) Environmental effects on protein glycosylation. Bio/Technology 8, 421–427.
Hayter, P.M., Curling, E.M.A., Baines, A.J., Jenkins, N., Salmon, I., Strange, P.G. and Bull A.T. (1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl. Microbiol. Biotechnol. 34, 559–564.
Hayter, P.M., Curling, E.M.A., Baines, A.J., Jenkins, N., Salmon, I., Strange, P.G., Tong, J.M. and Bull, A.T. (1992) Glucose limited chemostat culture of CHO cells producing recombinant interferon-γ. Biotechnol. Bioeng. 39, 327–335.
Kellerher, D.J., Kreibich, G. and Gilmore, R. (1992) Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein. Cell 69, 55–65.
Moonen, P., Mermod, J.J., Ernst, J.F., Hirschi, M., and DeLamarter, J.F. (1987) Increased biological activity of deglycosylated recombinant human granulocyte macrophage colony-stimulating factor produced by yeast or animal cells. Proc. Nat. Acad. Sci. 84, 4428–4431.
Parekh, R.B., Dwek, R.A., Thomas, J.R. Opdenakker, G., and Rademacher,T.W. (1989) Celltype-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator. Biochem. 28, 7644–7662.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1993 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Jenkins, N. et al. (1993). Changes in the Glycosylation of Interferon-γ during Culture. In: Kaminogawa, S., Ametani, A., Hachimura, S. (eds) Animal Cell Technology: Basic & Applied Aspects. Animal Cell Technology: Basic & Applied Aspects, vol 5. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-2044-9_32
Download citation
DOI: https://doi.org/10.1007/978-94-011-2044-9_32
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-4905-4
Online ISBN: 978-94-011-2044-9
eBook Packages: Springer Book Archive