Metabolic engineering of recombinant plasminogen sialylation in animal cells by tetracycline-regulated expression of murine CMP-NeuAc synthetase and hamster CMP-NeuAc transporter genes

Baker, K.N. and Johnson, I.D. and Roberts, G. and Cook, A. and Baines, Anthony J. and Gerardy-Schahn, R. and James, D.C. (1999) Metabolic engineering of recombinant plasminogen sialylation in animal cells by tetracycline-regulated expression of murine CMP-NeuAc synthetase and hamster CMP-NeuAc transporter genes. In: Bernard, A. and Griffiths, B. and Noe, W. and Wurm, F., eds. Animal Cell Technology: Products from Cells, Cells as Products. ESACT Meeting. Springer Netherlands, Netherlands pp. 263-265. ISBN 0-7923-6075-3. (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1007/0-306-46875-1_60

Abstract

It has been well documented that the degree of sialylation can affect the pharmacokinetics of a potential therapeutic product1. To date, the majority of efforts to augment sialylation have concentrated on expression of recombinant sialyltransferases in CHO and BHK cell lines, demonstrating variable results2–4. As there is now strong evidence to suggest that the availability of nucleotide-sugar substrate (CMP-NeuAc) in the Golgi lumen is an important factor limiting the rate of N-glycan sialylation5–6, we have targeted this metabolic control of sialylation. Our aim is to increase the rate of nucleotide sugar synthesis and transport into the Golgi lumen by coexpression of the recently cloned CMP-NeuAc synthetase7 and CMP-NeuAc transporter8 genes. Although high levels of gene expression are desirable to increase sialylation, these proteins may be lethal to the cell if over-expressed. To overcome this problem, a CHO cell line expressing a recombinant human plasminogen variant9 with well characterised N- and O-linked glycosylation sites10–13, was transfected with 5’ FLAG-tagged murine CMP-NeuAc synthetase or 3’ HA-tagged CMP-NeuAc transporter genes. Stable cell lines were produced with these genes under the control of a tetracycline-repressed promotor using the Tet-Off expression system (Clontech). The tetracycline (Tet)-regulated expression of a glycosyltransferase has previously been demonstrated as an effective means of altering the glycosylation of an engineered humanised IgG molecule14. Mutants of both genes have also been transfected into the tetracycline-regulated CHO cell line expressing the recombinant plasminogen variant to provide “inactive” negative controls. Thus, the level of expression of either or both genes can be varied to assess the degree of recombinant plasminogen sialylation in relation to the level of synthetase and/or transporter gene expression. Furthermore, expression can be tailored to control sialylation while minimising toxicity of the gene products to the cell.

Item Type: Conference or workshop item (Paper)
Subjects: Q Science
Q Science > QR Microbiology
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: M. Nasiriavanaki
Date Deposited: 24 Jul 2009 07:59
Last Modified: 17 Apr 2014 12:59
Resource URI: http://kar.kent.ac.uk/id/eprint/17035 (The current URI for this page, for reference purposes)
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