Cell-free methods to engineer therapeutic proteins : improving the efficacy of epidermal growth factor

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Protein-protein interactions such as ligand-receptor interactions mediate cell signaling and can be used to control and modulate cell behavior. Despite their effectiveness in natural settings, protein ligands require optimization of properties such as homogeneity, lack of immunogenicity, potency, or stability to be used in therapeutic applications. Multiple combinatorial and rational protein engineering techniques have been developed to improve therapeutic proteins. However, these methods have been limited by the lack of a robust, high-throughput system for the expression of complex mammalian proteins. In this dissertation, cell-free protein synthesis (CFPS) was applied to fill this void, and the potential of this approach was demonstrated with epidermal growth factor (EGF). EGF binds to the EGF receptor (EGFR) and stimulates cell proliferation, migration, and differentiation. EGF has been pursued for wound healing and tissue engineering applications, thus EGF mutants with enhanced biochemical and biophysical properties are of great biomedical interest. The primary goal of this thesis research was to create a directed evolution platform to screen protein libraries based on biological function, an objective not feasible with other protein engineering approaches. We developed methods to fully express mutated protein libraries and to determine the efficacy of each individual member by employing a high-throughput protein product assay followed by a cell-based functional assay conducted with uniform dosage. Using this platform, we discovered the first known EGF mutants with enhanced mitogenic activity compared to wild-type EGF despite many previous attempts. These mutants bound EGFR with approximately ten-fold weaker binding affinity than wild-type EGF and reduced depletion of soluble EGF and cell surface EGFR when cells were treated with EGF. Second, we employed CFPS to rapidly characterize an array of EGF mutants and rationally engineered mutants with ideal biochemical properties for EGFR activation. It is important to note that all of these studies can be carried out without the need for protein purification from the CFPS reaction mixture, significantly increasing the number of mutants that can be analyzed. Finally, we site-specifically incorporated non-natural amino acids into EGF by CFPS and demonstrated accessibility by conjugating EGF to polyethylene glycol (PEG), a polymer that has been shown to improve protein retention and stability. These improvements to EGF highlight the advantages of cell-free methods compared to other protein engineering strategies, and advance EGF's potential for therapeutic use. Furthermore, the techniques developed here can be extended to improve the biological efficacy of other clinically-relevant proteins.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Copyright date 2011
Publication date 2010, c2011; 2010
Issuance monographic
Language English


Associated with Lui, Bertrand Howyen
Associated with Stanford University, Department of Bioengineering.
Primary advisor Cochran, Jennifer R
Primary advisor Swartz, James R
Thesis advisor Cochran, Jennifer R
Thesis advisor Swartz, James R
Thesis advisor Quake, Stephen Ronald
Advisor Quake, Stephen Ronald


Genre Theses

Bibliographic information

Statement of responsibility Bertrand Howyen Lui.
Note Submitted to the Department of Bioengineering.
Thesis Thesis (Ph.D.)--Stanford University, 2011.
Location electronic resource

Access conditions

© 2011 by Bertrand Howyen Lui
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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