Date of Award
5-2024
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Bioengineering
Committee Chair/Advisor
David Karig, Ph.D.
Committee Member
Sarah Harcum, Ph.D.
Committee Member
Angela Alexander-Bryant, Ph.D.
Abstract
Centralized protein manufacturing platforms make the delivery of needed therapies to places with limited infrastructure almost impossible. Cell-free protein expression systems, systems that utilize protein production machinery extracted from cells, offer these communities a viable protein expression platform that is robust and easily deliverable to the place of need. Much work in cell-free system engineering looks to increase the hardiness of cell-free system (CFS) components, like the extract and reaction buffer, needed to carry transcription and translation of gene therapeutic targets forward. Freeze- and air-drying of extract, reaction buffer, or both with certain additives, like sugar molecules, has been proposed with some success. However, when CFS components are lyophilized or air-dried together, the system’s efficacy is significantly impacted when preserved over time and at non-ambient temperatures, which these systems will likely encounter in the field. More importantly, no methods have been proposed to improve cell-free system capacity outside standard reaction conditions. In other words, the operating range of cell-free systems is severely limited and needs to be addressed. Therefore, the purpose of this thesis work was two-fold. First, expand the operating range of cell-free systems and, second, better preserve CFS components.
To do so, a new experimental scheme was developed, namely the I3 scheme – identify, investigate, and integrate. First, climate-specific chaperones from extremophiles were identified from the literature in addition to gene regulatory elements controlling the chaperone’s expression in the CFS. Second, these chaperones were investigated in CFSs to gauge whether they support CFS capacity over a wide operating range, specifically high and low reaction temperatures. Third, these chaperones were integrated into the cell extract to elucidate their function in preserved and reacted CFSs.
These thesis results demonstrate that these CFS additives expand the operating range of CFSs relative to reaction temperature when expressed in parallel with the CFS or the cell extract. Additionally, it is demonstrated that these chaperones better preserve CFS components during air-drying and storage for up to 4 weeks. Together, these findings show that heterologous chaperones can function synergistically in CFSs. This work adds to the library of components at our disposal to make possible fieldable protein expression systems.
Recommended Citation
Becker, Matthew, "Broadening the Application Range of Cell-Free Protein Expression Systems" (2024). All Theses. 4218.
https://tigerprints.clemson.edu/all_theses/4218
Author ORCID Identifier
0009-0004-7566-7501