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Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome

Lookup NU author(s): Professor Phillip WrightORCiD

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Abstract

© 2017 Elsevier Inc. 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%.


Publication metadata

Author(s): Strutton B, Jaffe SRP, Pandhal J, Wright PC

Publication type: Article

Publication status: Published

Journal: Biochemical and Biophysical Research Communications

Year: 2018

Volume: 495

Issue: 1

Pages: 686-692

Print publication date: 01/01/2018

Online publication date: 04/11/2017

Acceptance date: 03/11/2017

ISSN (print): 0006-291X

ISSN (electronic): 1090-2104

Publisher: Elsevier BV

URL: https://doi.org/10.1016/j.bbrc.2017.11.023

DOI: 10.1016/j.bbrc.2017.11.023


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