Protein engineering can be made far more powerful if a protein is not only expressed recombinantly but also altered covalently using synthetic chemistry. These two methods are brought together in the protein semi-synthesis technique Expressed Protein Ligation (EPL). In EPL, recombinant and synthetic polypeptides are joined together via a chemoselective ligation reaction. EPL was originally used to attach synthetic constructs to the C-terminus of recombinant proteins, but is now used to attach recombinant or synthetic polypeptides either at the N- or C-terminus of a protein or into the core of a protein. This thesis illustrates, with three distinct applications, the development of EPL from its original definition to its current understanding. In the first application, a general strategy was developed for the site-specific incorporation of fluorophores into proteins using Abl-SH3 as a model system. In the second application, chemistries were developed that allowed the site-specific introduction of phospho-amino acids into proteins, in this case using the transforming growth factor β receptor I as the model system. In the final application, EPL was used to synthesize several modified versions of the E. coli sigma factor σ70, demonstrating that this method can be used to probe extremely large macromolecules. These studies revealed that EPL works under a variety of reaction conditions and provided paradigms for using this technique to site-specifically insert fluorophores and phosphate groups into proteins. The chemical manipulation of proteins by EPL will be an important tool as researchers strive to characterize the proteomes of organisms.
","abstract_html":"<p>Protein engineering can be made far more powerful if a protein is not only expressed recombinantly but also altered covalently using synthetic chemistry. These two methods are brought together in the protein semi-synthesis technique Expressed Protein Ligation (EPL). In EPL, recombinant and synthetic polypeptides are joined together via a chemoselective ligation reaction. EPL was originally used to attach synthetic constructs to the C-terminus of recombinant proteins, but is now used to attach recombinant or synthetic polypeptides either at the N- or C-terminus of a protein or into the core of a protein. This thesis illustrates, with three distinct applications, the development of EPL from its original definition to its current understanding. In the first application, a general strategy was developed for the site-specific incorporation of fluorophores into proteins using Abl-SH3 as a model system. In the second application, chemistries were developed that allowed the site-specific introduction of phospho-amino acids into proteins, in this case using the transforming growth factor β receptor I as the model system. In the final application, EPL was used to synthesize several modified versions of the E. coli sigma factor σ<sup>70</sup>, demonstrating that this method can be used to probe extremely large macromolecules. These studies revealed that EPL works under a variety of reaction conditions and provided paradigms for using this technique to site-specifically insert fluorophores and phosphate groups into proteins. The chemical manipulation of proteins by EPL will be an important tool as researchers strive to characterize the proteomes of organisms.</p>","abstract_has_math":false,"creators":["Holford, Mande"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Tom Muir"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2002,"date_issued":"2002-01-01T08:00:00Z","date_published":"2002-01-01T08:00:00Z","updated_at":"2026-07-24T04:11:51Z","subjects":["protein semi-synthesis","expressed protein ligation","site-specific labeling","phospho-amino acids","sigma factors","protein engineering","Life Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/382","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Tom Muir"]},{"key":"dc:creator","label":"Author","values":["Holford, Mande"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["protein semi-synthesis","expressed protein ligation","site-specific labeling","phospho-amino acids","sigma factors","protein engineering","Life Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/382"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Protein engineering can be made far more powerful if a protein is not only expressed recombinantly but also altered covalently using synthetic chemistry. These two methods are brought together in the protein semi-synthesis technique Expressed Protein Ligation (EPL). In EPL, recombinant and synthetic polypeptides are joined together via a chemoselective ligation reaction. EPL was originally used to attach synthetic constructs to the C-terminus of recombinant proteins, but is now used to attach recombinant or synthetic polypeptides either at the N- or C-terminus of a protein or into the core of a protein. This thesis illustrates, with three distinct applications, the development of EPL from its original definition to its current understanding. In the first application, a general strategy was developed for the site-specific incorporation of fluorophores into proteins using Abl-SH3 as a model system. In the second application, chemistries were developed that allowed the site-specific introduction of phospho-amino acids into proteins, in this case using the transforming growth factor β receptor I as the model system. In the final application, EPL was used to synthesize several modified versions of the E. coli sigma factor σ70, demonstrating that this method can be used to probe extremely large macromolecules. These studies revealed that EPL works under a variety of reaction conditions and provided paradigms for using this technique to site-specifically insert fluorophores and phosphate groups into proteins. The chemical manipulation of proteins by EPL will be an important tool as researchers strive to characterize the proteomes of organisms.
"]},{"key":"dc:title","label":"Title","values":["Site-Specific Incorporation of Biochemical and Biophysical Probes into Proteins Using Expressed Protein Ligation"]}]}],"canonical_facts":{"dc:contributor":["Tom Muir"],"dc:creator":["Holford, Mande"],"dc:description.abstract":["Protein engineering can be made far more powerful if a protein is not only expressed recombinantly but also altered covalently using synthetic chemistry. These two methods are brought together in the protein semi-synthesis technique Expressed Protein Ligation (EPL). In EPL, recombinant and synthetic polypeptides are joined together via a chemoselective ligation reaction. EPL was originally used to attach synthetic constructs to the C-terminus of recombinant proteins, but is now used to attach recombinant or synthetic polypeptides either at the N- or C-terminus of a protein or into the core of a protein. This thesis illustrates, with three distinct applications, the development of EPL from its original definition to its current understanding. In the first application, a general strategy was developed for the site-specific incorporation of fluorophores into proteins using Abl-SH3 as a model system. In the second application, chemistries were developed that allowed the site-specific introduction of phospho-amino acids into proteins, in this case using the transforming growth factor β receptor I as the model system. In the final application, EPL was used to synthesize several modified versions of the E. coli sigma factor σ70, demonstrating that this method can be used to probe extremely large macromolecules. These studies revealed that EPL works under a variety of reaction conditions and provided paradigms for using this technique to site-specifically insert fluorophores and phosphate groups into proteins. The chemical manipulation of proteins by EPL will be an important tool as researchers strive to characterize the proteomes of organisms.
"],"dc:identifier":["https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/382"],"dc:subject":["protein semi-synthesis","expressed protein ligation","site-specific labeling","phospho-amino acids","sigma factors","protein engineering","Life Sciences"],"dc:title":["Site-Specific Incorporation of Biochemical and Biophysical Probes into Proteins Using Expressed Protein Ligation"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T04:11:51Z"}