IMPROVING GLYCOSYLATION EFFICIENCY IN ESCHERICHIA COLI BY MAMMALIAN-CELL-INSPIRED PROTEIN-PROTEIN INTERACTIONS

Asparagine linked glycosylation (N-glycosylation) is one of the most common post-translational modifications found in proteins. The central enzyme in the N-glycosylation pathway responsible for the transfer of glycans onto asparagine residues is called the oligosaccharyltransferase (OST). In mammalian cells, most proteins are N-glycosylated cotranslationally, mediated by protein-protein interactions between the OST and the general secretion (Sec) pathway. This mechanism is important for efficient glycosylation of many structured proteins. Recombinant N-glycosylation in Escherichia coli also shows a dependence on the structural context of the acceptor protein. However, intentional coordination between protein secretion and glycosylation in this engineered system has yet to be demonstrated. Here we investigated two strategies using artificial protein-protein interactions to couple the protein secretion and glycosylation machinery in E. coli. A novel model acceptor protein based on the human prosaposin was developed. While the proposed designs were ineffective under the conditions tested, we discovered that fusion proteins of the Campylobacter jejuni PglB were able to improve the expression of challenging acceptor proteins without sacrificing glycosylation efficiency. Further investigations reveal significant implications relating OST expression to protein glycosylation efficiency.

Name thesis:degree_name

M.S., Chemical Engineering

Level thesis:degree_level

Master of Science

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

Cornell University

Year dc:date.issued

2023

Author dc:creator

• Chen, Jiazhen

Committee member dc:contributor.committeemember

• Li, Sijin

Language dc:language.iso

en

DOI

https://doi.org/10.7298/9qff-za59

Dc Identifier Other

ProQuest Submission ID: 11912
ProQuest Publication ID: 30576144

OAI identifier oai:identifier

oai:ecommons.cornell.edu:1813/114520