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Induction of protein aggregation in an early secretory compartment by elevation of expression level

Schröder, M.; Schäfer, R.; Friedl, P.

Authors

R. Schäfer

P. Friedl



Abstract

A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate-limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide-bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H-sensitivity and located to the endoplasmic reticulum and the cis-Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D-glucose and L-glutamine, on the ATIII-yield. We found that utilization of D-glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII-synthesized per L-glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L-glutamine.

Citation

Schröder, M., Schäfer, R., & Friedl, P. (2002). Induction of protein aggregation in an early secretory compartment by elevation of expression level. Biotechnology and Bioengineering, 78(2), 131-140. https://doi.org/10.1002/bit.10206

Journal Article Type Article
Publication Date Apr 1, 2002
Deposit Date May 17, 2007
Journal Biotechnology and Bioengineering
Print ISSN 0006-3592
Electronic ISSN 1097-0290
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 78
Issue 2
Pages 131-140
DOI https://doi.org/10.1002/bit.10206
Keywords Unfolded protein response, Endoplasmic reticulum, Aggregation, Recombinant protein production.
Publisher URL http://www3.interscience.wiley.com/cgi-bin/abstract/90511184/ABSTRACT