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

Author

Mande Holford

Date of Award

2002

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

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.

Comments

Submitted in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Holford, Mande, "Site-Specific Incorporation of Biochemical and Biophysical Probes into Proteins Using Expressed Protein Ligation" (2002). Student Theses and Dissertations. 382.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/382