ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE
| dc.contributor.author | Kwon, Yong Hyun | |
| dc.contributor.chair | DeLisa, Matthew | |
| dc.contributor.committeeMember | Daniel, Susan | |
| dc.date.accessioned | 2021-12-20T20:34:30Z | |
| dc.date.available | 2021-12-20T20:34:30Z | |
| dc.date.issued | 2021-08 | |
| dc.description | 49 pages | |
| dc.description.abstract | Oligosaccharyltransferase (OST) is a key enzyme in the asparagine-linked (N-linked) protein glycosylation pathway. OSTs exist in all domains of life and are capable of transferring a preassembled glycan from lipid carrier to an acceptor peptide. Bacterial OSTs are an single-subunit enzyme that are amenable to recombinant expression in model organism including Escherichia coli. As a result, bacterial OSTs have been used as models to explore the mechanism of the N-linked glycosylation process in nature. These developments, notwithstanding, recombinant expression and purification of the OST enzymes remain significant challenges. Bacterial OSTs are multi-pass transmembrane protein that requires intricate balance between protein synthesis rate and a pace of membrane insertion. Further, membrane protein purification often necessitates the use of ultracentrifugation and detergent, both of which limit process scalability and compatibility. To address these challenges, we proposed a protein engineering strategy called SIMPLEx or solubilization of integral membrane proteins with high levels of expression to generate water-soluble variants of the bacterial OST. Specifically, we designed several OST chimeras where the N-terminus of the OST is fused with the amphipathic protein including engineered human apolipoprotein A-I. Using E. coli culture as an expression platform, several SIMPLEx-OSTs could be expressed within the cytoplasmic fraction of the E. coli. Importantly, our engineered OSTs retain their biological activity and are able to N-glycosylate several acceptor proteins including therapeutic human growth hormone. Collectively, our OST-engineering strategy is anticipated to generate a new subclass of water-soluble N-OST enzymes with applications in bioproduction of the glycotherapeutics and glycovaccines. | |
| dc.identifier.doi | https://doi.org/10.7298/jx5a-q006 | |
| dc.identifier.other | Kwon_cornell_0058O_11270 | |
| dc.identifier.other | http://dissertations.umi.com/cornell:11270 | |
| dc.identifier.uri | https://hdl.handle.net/1813/110422 | |
| dc.language.iso | en | |
| dc.subject | Glyco | |
| dc.subject | membrane | |
| dc.subject | Oligosaccharyltransferase | |
| dc.subject | PglB | |
| dc.title | ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE | |
| dc.type | dissertation or thesis | |
| dcterms.license | https://hdl.handle.net/1813/59810 | |
| thesis.degree.discipline | Chemical Engineering | |
| thesis.degree.grantor | Cornell University | |
| thesis.degree.level | Master of Science | |
| thesis.degree.name | M.S., Chemical Engineering |
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