2026-09-19T18:50:10Zhttps://ecommons.cornell.edu/server/oai/requestoai:ecommons.cornell.edu:1813/1104222026-05-15T19:43:46Zcom_1813_35col_1813_47
Kwon, Yong Hyun
DeLisa, Matthew
Daniel, Susan
2021-12-20T20:34:30Z
2021-12-20T20:34:30Z
2021-08
ProQuest Submission ID: 11270
ProQuest Publication ID: 28652681
https://hdl.handle.net/1813/110422
https://doi.org/10.7298/jx5a-q006
15160304
49 pages
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.
en
Glyco
membrane
Oligosaccharyltransferase
PglB
ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE
dissertation or thesis
https://newcatalog.library.cornell.edu/catalog/15160304
application/pdf
Chemical Engineering
Cornell University
Master of Science
M.S., Chemical Engineering
https://hdl.handle.net/1813/59810
Publication
Cornell Theses and Dissertations
Kwon, Yong Hyun
5893a6ea-7af3-41d7-abc6-04bcd26ab5df
open.access
open.access
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<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.</Abstract>
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