Site-Specific Incorporation of Biochemical and Biophysical Probes into Proteins Using Expressed Protein Ligation

<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 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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2002

Author dc:creator

• Holford, Mande

Contributors dc:contributor

• Tom Muir

• protein semi-synthesis
• expressed protein ligation
• site-specific labeling
• phospho-amino acids
• sigma factors
• protein engineering
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/382

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1389