Strutton, B., Jaffé, S.R.P., Pandhal, J. et al. (1 more author) (2018) Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome. Biochemical and Biophysical Research Communications, 495 (1). pp. 686-692. ISSN 0006-291X
Abstract
Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2017 Elsevier |
Keywords: | Bacterial N-linked glycosylation; Glycoprotein producing host strain; Escherichia coli; Oligosaccharyl transferase; Glycosylation efficiency; Absolute quantification glycoprotein |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL (BBSRC) BB/K011200/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 01 Mar 2018 13:18 |
Last Modified: | 01 Mar 2018 13:18 |
Published Version: | https://doi.org/10.1016/j.bbrc.2017.11.023 |
Status: | Published |
Publisher: | Elsevier |
Refereed: | Yes |
Identification Number: | 10.1016/j.bbrc.2017.11.023 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:127946 |