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Title: Engineering Escherichia coli to improve its N-linked glycosylation capabilities and the development of a new method of quantifying production
Author: Strutton, Benjamin
ISNI:       0000 0004 6062 5221
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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The field of recombinant glycoprotein production in Escherichia coli has advanced with an increase in the number of glycan structures that can be transferred to target proteins (Schwarz et al. 2010, Valderrama-Rincon et al. 2012, Wetter et al. 2013, Srichaisupakit et al. 2015). With various industrial and academic groups utilising a variety of the available structures, the need for a host strain of E. coli that can transfer a number of different glycans is evident. Here the bacterial oligosaccharyl transferase, pglB, known to transfer glycans of alternate content and structure, was chromosomally located creating an E. coli glycosylation host strain where the target protein, glycosyltransferases, and sugar synthesis genes could be switched to produce a glycoprotein of choice. To assess the glycoprotein producing capacity of the newly developed strain, a combined mass spectrometry and Western blot approach was developed to enable absolute quantification of target protein production. With pglB located on the chromosome glycosylation efficiency rose 85% with a 17% increase in glycoprotein production. In an attempt to further improve the efficiency of glycosylation, which is affected with the incorporation of glycosyltransferases from alternate organisms, a multitude of genes identified from previous exploratory studies were over expressed in combination. Certain combinations improved the glycosylation process with higher efficiency and a greater percentage of the desired diglycosylated product, although one gene, dxs, was found to hinder production. It is expected that the creation of the platform glycosylating strain of E. coli will enable users to more easily test and create novel recombinant glycoproteins. Alongside this the development of the combined mass spectrometry and Western approach will allow absolute quantification of the produced glycoforms, pushing towards a move away from purely Western blot analysis and relative protein production.
Supervisor: Wright, Phillip ; Pandhal, Jagroop Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available