{"id":{"repo_id":"cornell","oai_identifier":"oai:ecommons.cornell.edu:1813/110422"},"canonical_url":"https://search.dev.ndltd.org/etd/cornell/oai:ecommons.cornell.edu:1813/110422","repository":{"repo_id":"cornell","name":"Cornell University","base_url":"https://ecommons.cornell.edu/server/oai/request"},"display":{"title":"ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE","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_html":"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_has_math":false,"creators":["Kwon, Yong Hyun"],"institution":"Cornell University","degree_name":"M.S., Chemical Engineering","degree_level":"Master of Science","degree_discipline":"Chemical Engineering","degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":["Daniel, Susan"],"year":2021,"date_issued":"2021-08","date_published":"2021-08","updated_at":"2026-07-24T01:49:00Z","subjects":["Glyco","membrane","Oligosaccharyltransferase","PglB"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7298/jx5a-q006"],"render_values":[{"text":"https://doi.org/10.7298/jx5a-q006","href":"https://doi.org/10.7298/jx5a-q006","code":true}]},{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["ProQuest Submission ID: 11270","ProQuest Publication ID: 28652681"],"render_values":[{"text":"ProQuest Submission ID: 11270","href":null,"code":true},{"text":"ProQuest Publication ID: 28652681","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/1813/110422","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Daniel, Susan"]},{"key":"dc:creator","label":"Author","values":["Kwon, Yong Hyun"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2021-12-20T20:34:30Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2021-12-20T20:34:30Z"]},{"key":"dc:date.issued","label":"Date","values":["2021-08"]},{"key":"dc:type","label":"Dc Type","values":["dissertation or thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemical Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Master of Science"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M.S., Chemical Engineering"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Cornell University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Glyco","membrane","Oligosaccharyltransferase","PglB"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7298/jx5a-q006"]},{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["ProQuest Submission ID: 11270","ProQuest Publication ID: 28652681"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/1813/110422"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["49 pages"]},{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE"]}]}],"canonical_facts":{"dc:contributor.committeemember":["Daniel, Susan"],"dc:creator":["Kwon, Yong Hyun"],"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:format.mimetype":["application/pdf"],"dc:identifier.doi":["https://doi.org/10.7298/jx5a-q006"],"dc:identifier.other":["ProQuest Submission ID: 11270","ProQuest Publication ID: 28652681"],"dc:identifier.uri":["https://hdl.handle.net/1813/110422"],"dc:language.iso":["en"],"dc:subject":["Glyco","membrane","Oligosaccharyltransferase","PglB"],"dc:title":["ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE"],"dc:type":["dissertation or thesis"],"thesis:degree_discipline":["Chemical Engineering"],"thesis:degree_level":["Master of Science"],"thesis:degree_name":["M.S., Chemical Engineering"],"thesis:institution_name":["Cornell University"]},"updated_at":"2026-07-24T01:49:00Z"}