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Ostwalt, Alexis Daniel, Susan Baskin, Jeremy Ober, Christopher 2025-06-30T22:03:52Z 2025-06-30T22:03:52Z 2024-12 ProQuest Submission ID: 14669 ProQuest Publication ID: 31563030 https://hdl.handle.net/1813/117221 http://doi.org/10.7298/tfaw-mn19 16921966 148 pages In biotechnology, the study of membrane proteins is of high interest due to their unique physical properties used as invaluable tools for the development of new therapeutics. However, honing down on the structural and functional properties of membrane proteins is no easy feat, owing to challenges in expression, purification, and the availability of high-throughput functional readout methods. Historically, cell-based protein production methods have dominated the field of membrane protein studies; but now, cell-free protein synthesis (CFPS) approaches are beginning to both complement and rival traditional expression methods. Regarding readout methods, maintaining the structural and functional properties of membrane proteins requires an easily controllable and specific lipid environment that promotes lateral diffusion and protein-lipid interactions. Here, I present a customizable cell-free platform for the functional assembly and integration of membrane proteins in both liposomes and supported lipid bilayers (SLBs) to qualitatively assess membrane protein activity. Our platform merges the convenience and scalability of CFPS with two functional readout methods: a fluorescence assay in liposomes and an SLB bioelectronic platform. With these tools, we can directly detect the activity of difficult-to-study membrane proteins like intracellular ion transporters. Additionally, we can further characterize membrane protein expression, orientation, and function in an isolated and controllable environment. With the momentum of developing improved CFPS and biosensing technologies, this platform can aid in extending the limits of membrane protein biotechnology. en Attribution-NoDerivatives 4.0 International https://creativecommons.org/licenses/by-nd/4.0/ cell-free lipids liposomes supported lipid bilayers transmembrane proteins Cell-free platform for the functional characterization of intracellular ion transporters dissertation or thesis https://newcatalog.library.cornell.edu/catalog/16921966 application/pdf Chemical Engineering Cornell University Doctor of Philosophy Ph. D., Chemical Engineering https://hdl.handle.net/1813/59810.2 Publication Cornell Theses and Dissertations Ostwalt, Alexis 5893a6ea-7af3-41d7-abc6-04bcd26ab5df open.access open.access <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="04a684cd-9d47-4be5-b0a9-b4f8fc7fb069"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>en</Language> <Title>Cell-free platform for the functional characterization of intracellular ion transporters</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2024-12</PublicationDate> <DOI>http://doi.org/10.7298/tfaw-mn19</DOI> <Authors> <Author> <DisplayName>Ostwalt, Alexis</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <OrgUnit /> </Publisher> </Publishers> <License>https://creativecommons.org/licenses/by-nd/4.0/</License> <Keyword>cell-free</Keyword> <Keyword>lipids</Keyword> <Keyword>liposomes</Keyword> <Keyword>supported lipid bilayers</Keyword> <Keyword>transmembrane proteins</Keyword> <Abstract>In biotechnology, the study of membrane proteins is of high interest due to their unique physical properties used as invaluable tools for the development of new therapeutics. However, honing down on the structural and functional properties of membrane proteins is no easy feat, owing to challenges in expression, purification, and the availability of high-throughput functional readout methods. Historically, cell-based protein production methods have dominated the field of membrane protein studies; but now, cell-free protein synthesis (CFPS) approaches are beginning to both complement and rival traditional expression methods. Regarding readout methods, maintaining the structural and functional properties of membrane proteins requires an easily controllable and specific lipid environment that promotes lateral diffusion and protein-lipid interactions. Here, I present a customizable cell-free platform for the functional assembly and integration of membrane proteins in both liposomes and supported lipid bilayers (SLBs) to qualitatively assess membrane protein activity. Our platform merges the convenience and scalability of CFPS with two functional readout methods: a fluorescence assay in liposomes and an SLB bioelectronic platform. With these tools, we can directly detect the activity of difficult-to-study membrane proteins like intracellular ion transporters. Additionally, we can further characterize membrane protein expression, orientation, and function in an isolated and controllable environment. With the momentum of developing improved CFPS and biosensing technologies, this platform can aid in extending the limits of membrane protein biotechnology.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>