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Browsing by Subject "Propensity"

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    2018 Undergraduate Research Symposium Presentation on Method for Detection for Protein Aggregation Propensity
    (2018) Fransen, Katharina A; Ritter, Seth C; Hackel, Benjamin
    Engineered proteins are valuable tools for clinical therapeutics and diagnostics as well as for other biotechnology applications. The physicochemical robustness of a protein is an important factor in its utility. For example, protein solubility is advantageous for production, conjugation, formulation, and use; yet numerous engineered proteins exhibit suboptimal solubility due to the formation of protein aggregates. For the technological aim of identifying superior reagents and the scientific goal of elucidating protein sequence – function relationships, we are developing a method for high-throughput analysis of protein aggregation. The method uses yeast surface display in which the protein of interest (POI) is coupled to the surface of a S. cerevisiae yeast cell via an extended ‘PAS40’ polypeptide linker to the native yeast mating protein Aga2p, which binds to the yeast-bound Aga1 membrane protein. Meanwhile, the DNA encoding the POI is retained inside the yeast thereby creating a phenotype-genotype linkage that enables independent evaluation of millions of POI variants with simple DNA sequencing to reveal the identity of functional variants after aggregation analysis. Aggregation analysis utilizes Foerster resonance energy transfer (FRET) in which an excited donor fluorophore transfers energy to a nearby acceptor fluorophore. The acceptor and donor fluorophores are attached near the N-terminus of the POIs, allowing aggregating POIs to bring the fluorophores within the necessary radius for energy transfer and subsequent acceptor fluorescence. Measurement of the fluorescence of the acceptor protein allows for the aggregation analysis of the POI, which can be performed in high-throughput (100 million per hour) via flow cytometry.