Directed Evolution of Protein Receptor Binding for Small Molecule Therapeutics Using Fluorescence Polarization

• Main Author: Bannier, Sean David
• Language: English
• Published: 2021
• Subjects: Chemistry; Biochemistry; Biophysics; Tetracylcines; Protein binding; Polarization (Light); Yeast
• Online Access: https://doi.org/10.7916/d8-v1hm-cd27

The field of metabolic engineering focuses on using molecular biology tools to genetically modify the metabolic pathways of cells for the production of chemical compounds. The field of directed evolution can alter the native abilities of proteins by taking inspiration from natural evolution. Both fields bring novel solutions to current problems in energy, the environment, and medicine. However, there is still no general higher throughput screening method for both of these fields. In this dissertation, we apply our designed fluorescence polarization assay to fill this need in the fields of metabolic engineering and directed evolution. Chapter 0 gives background information related to metabolic engineering, directed evolution, tetracyclines, the Tetracycline Repressor protein (TetR), TAN-1612, and fluorescence polarization. Chapter 1 describes our development of a quantitive, sensitive, and fast fluorescence polarization assay which uses the TetR protein to detect the binding of the small molecule tetracycline TAN-1612. Chapter 2 demonstrates that the binding affinity of the TetR protein for TAN-1612 can be improved using directed evolution and by incorporating our assay to screen TetR mutants. Finally, in Chapter 3 we apply our fluorescence polarization assay to the screening of yeast strains biosynthesizing TAN-1612, without the need of time and labor intensive extraction and purification steps.