Protein Engineering for Biosensor Development

<p>Biosensors incorporating proteins as molecular recognition elements for analytes are used in clinical diagnostics, as biological research tools, and to detect chemical threats and pollutants. This work describes the application of protein engineering techniques to address three aspects in the design of protein-based biosensors; the transduction of binding into an observable, the manipulation of affinities, and the diversification of specificities. The periplasmic glucose-binding protein from the hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein variants to construct a fluorescent ratiometric sensor that is sufficiently robust to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by altering a C-terminal helical domain that tunes ligand binding affinity through conformational coupling effects. This method was extended to the Escherichia coli arabinose-binding protein. Computational design techniques were used to diversify the specificity of the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions that control conformational coupling. These experiments demonstrate that long-range conformational effects as well as molecular recognition interactions need to be considered in the design of high-affinity receptors.</p>Dissertatio