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dc.contributor.advisorBaker, David
dc.contributor.advisorKlavins, Eric
dc.contributor.authorYounger, David Aaron
dc.date.accessioned2017-10-26T20:47:09Z
dc.date.submitted2017-07
dc.identifier.otherYounger_washington_0250E_17710.pdf
dc.identifier.urihttp://hdl.handle.net/1773/40488
dc.descriptionThesis (Ph.D.)--University of Washington, 2017-07
dc.description.abstractHigh-throughput methods for screening protein-protein interactions enable the rapid characterization of engineered binding proteins and interaction networks. While existing approaches are powerful, none allow quantitative library-on-library characterization of protein interactions in a modifiable extracellular environment. Here, we show that sexual agglutination of S. cerevisiae can be reprogrammed to link interaction strength with mating efficiency using synthetic agglutination (SynAg). Validation of SynAg with 89 previously characterized interactions shows a log-linear relationship between mating efficiency and protein binding strength for interactions with KD’s ranging from below 500 pM to above 300 μM. Using induced chromosomal translocation to pair barcodes representing binding proteins, thousands of distinct interactions can be screened in a single pot. We demonstrate the ability to characterize protein interaction networks in a modifiable environment by introducing a soluble peptide that selectively disrupts a subset of interactions in a representative network by up to 800-fold. SynAg enables the high-throughput, quantitative characterization of protein-protein interaction networks in a fully-defined extracellular environment at a library-on-library scale.
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.rightsCC BY-NC-SA
dc.subjectHigh-throughput screening
dc.subjectProtein Engineering
dc.subjectProtein-protein interaction networks
dc.subjectSynthetic Biology
dc.subjectYeast Mating
dc.subjectBiomedical engineering
dc.subjectBiochemistry
dc.subjectCellular biology
dc.subject.otherBioengineering
dc.titleHigh-Throughput Characterization of Protein-Protein Interactions by Reprogramming Yeast Mating
dc.typeThesis
dc.embargo.termsDelay release for 1 year -- then make Open Access
dc.embargo.lift2018-10-26T20:47:09Z


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