Use this URL to cite or link to this record in EThOS:
Title: Towards the cell-free expression of glycoproteins
Author: Exley, Kealan Peter
ISNI:       0000 0004 7963 7097
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
Availability of Full Text:
Access from EThOS:
Access from Institution:
N-linked glycosylation, is the covalent linkage of an oligosaccharide to an amido-group of an asparagine in a consensus sequence motif. The biosynthesis and transfer of a carbohydrate chain onto a nascent protein occurs within the ER and these initial steps are highly conserved throughout eukaryotes. Further processing of the carbohydrate chain occurs in the Golgi apparatus, where differences in processing between species and cell-types arise, resulting in different saccharide composition of the end-product. These differences are important in nature as N-linked glycosylation can influence a protein's function and interaction. However, this trait can be problematic when manufacturing a biopharmaceutical, due to the potential pharmacokinetic influence of the glycan structure. Consequently, it is important to ensure a suitable N-linked glycan is produced. The removal of the N-linked glycosylation processing from the constraints of a cell will allow for greater control of the end saccharide composition of the glycan chain. A cell-free protein expression system will reduce heterogeneity allowing for the production of a homogenesis glycan chain. With this in mind, a project was conceived to create a cell free protein synthesis system for the expression of glycoproteins. The project commenced with implementation of a glycoengineering approach on P. pastoris to generate pentamannosyl oligosaccharide. This oligosaccharide is seen as substrate for the synthesis of a complex-type glycoprofile. To process the pentamannosyl oligosaccharide the expression of a glycosidase and two glycotransferases was attempted. The feasibility of producing a cell-free protein synthesis (CFPS) system from yeast cell lysate was also tested. This project resulted in a knockout P. pastoris strain which prevented the hypermannosylation glycoprofile. This strain can viably be used to generate the pentamannosyl oligosaccharide. A recombinant human GlcNAcT-I was produced which demonstrated the in vitro modification of an oligosaccharide chain. Finally, the project developed an active CFPS with a S. cerevisiae cell lysate but not with P. pastoris, suggesting that not all cell lysates can be utilised to generate an active CFPS. Together, these results highlight the difficulties in utilising a non-conventional yeast for genetic and metabolic engineering. This research has suggested targets for future work which could yet see the generation the first cell free glycoprotein synthesis with a complex-type glycoprofile.
Supervisor: Polizzi, Karen ; Kontoravdi, Cleo Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral