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dc.contributor.advisorWall, Gerard
dc.contributor.authorO' Connor, Iain
dc.date.accessioned2016-11-14T11:39:32Z
dc.date.available2016-11-14T11:39:32Z
dc.date.issued2016-11-14
dc.identifier.urihttp://hdl.handle.net/10379/6158
dc.description.abstractRecombinant protein production (RPP) in Escherichia coli benefits from a long history of research which has facilitated the development of a plethora of strains and vectors suited to RPP applications. The identification and functional transfer to E. coli of an N-linked protein glycosylation system, pgl, from Campylobacter jejuni has recently enabled investigation of novel glycoprotein-based applications previously not possible in the organism. To date, a number of glyco-conjugate vaccines have been successfully produced in E. coli, and work towards “humanisation” of the characteristic C. jejuni glycan chain is continuing in order to produce glycoproteins suited to in vivo applications. Meanwhile, the C. jejuni glycan chain has been successfully attached to single-chain antibody fragments (scFvs) to mediate their covalent attachment to functionalised surfaces for improved immunosensor development. In this study, the pgl system was further engineered by mutagenic analysis of the glycosyltransferase PglJ – which is responsible for addition of the third sugar of the heptasaccharide N-glycan chain. Mutagenesis of E276 and E284 of the functionally important EX7E motif resulted in glycoprofiles indicating that glycan chain extension was not completely eliminated in either glycovariant, in contrast to published results with homologous glycosyltransferase enzymes. Inactivation of PglJ by termination of translation or by creation of a E276A/E284A double mutant completely eliminated glycosyltransferase activity of PglJ. Termination of translation of PglH, which further extends the glycan chain produced by PglJ, was also carried out to confirm the partial activity of the PglJ single-site mutants. Disaccharide-linked scFvs produced in E. coli pglJ- cells were compared to fully (heptasaccharide-containing)-glycosylated and unglycosylated scFvs in surface attachment and antigen-binding studies. Adsorption of all scFvs onto polystyrene occurred at similar rates whereas both glycosylated protein variants displayed a specific and more rapid attachment (up to five-fold greater coating) to amine-functionalised surfaces than their unglycosylated counterpart . Upon adsorption, the three scFv variants exhibited similar antigen binding while the covalently attached, glycosylated scFvs displayed up to eight-fold higher antigen binding than the unglycosylated scFvs. This demonstrated the potential application of the truncated glycan in protein immobilisation, while minimising its immunogenicity and potential susceptibility of the sugar chain to degradation by glycosidase enzymes in vivo. Comparison of adsorption and covalent attachment of scFvs to functionalised cardiovascular stent materials confirmed the efficacy of immobilising scFvs for such applications via the shortened glycan tag. Meanwhile, the use of atomic force microscopy provided insight into the spatial positioning and distribution of covalently-immobilised scFvs on the stent material. A lectin array analysis was carried out to query glycovariants produced by E. coli cells harbouring the engineered C. jejuni pgl constructs against a broad panel of carbohydrate-binding lectins. This rapid and multivariate typing investigation identified SBA as a candidate lectin that was used to distinguish between the glycovariant sequences in western blots. Efforts to extend the C. jejuni-pgl-encoded glycan chain through the action of the CgtB enzyme derived from the LOS locus of C. jejuni, identified a number of bottlenecks in cgtB expression in E. coli and P. pastoris. Potential solutions to express the relevant glycosyltransferases and mediate terminal capping of E. coli-produced glycoproteins with sialic acids were established for future investigations. These results illustrate the potential of expression of recombinant glycoproteins and their glycan engineering in E. coli in fundamental studies of glycosyltransferase enzymes and in biotechnological applications. Bioconjugates that exhibit improved immobilisation and antigen binding over their absorbed, non-glycosylated counterparts can be readily produced via the incorporation of a minimal recombinant tag. The study also establishes characterisation approaches to detect and analyse subtle glycan changes in recombinant glycoproteins. The C. jejuni pgl glycosylation system utilised in this work will likely see increased adoption and continued engineering in RPP in the near future and this study contributes to its ongoing adaptation for applications in fields such as diagnostics and therapeutics.en_IE
dc.subjectGlycosylationen_IE
dc.subjectEscherichia colien_IE
dc.subjectRecombinant Protein Productionen_IE
dc.subjectAntibodyen_IE
dc.subjectAntibody fragmenten_IE
dc.subjectscFven_IE
dc.subjectFluoresceinen_IE
dc.subjectImmobilisationen_IE
dc.subjectWestern bloten_IE
dc.subjectMicroscopyen_IE
dc.subjectCovalenten_IE
dc.subjectAdsorptionen_IE
dc.subjectPeriplasmen_IE
dc.subjectLectinen_IE
dc.subjectProtein purificationen_IE
dc.subjectMicrobiologyen_IE
dc.titleGlycosylation and glycoengineering of recombinant antibody fragments in Escherichia colien_IE
dc.typeThesisen_IE
dc.contributor.funderNUI Galway College of Scienceen_IE
dc.local.noteThe aim of this project was to produce a variety of glycosylated proteins in Escherichia coli. The mechanism for sugar coating proteins was exploited to produce alternative glycoproteins and these were subsequently validated and investigated for their potential in a range of applications.en_IE
dc.local.finalYesen_IE
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