Individuals in the U.S. with kidney conditions including infection, kidney stones, cancer, or missing kidney exceed 2.5 million annually. Most renal diseases affect serum urea concentration which provides a sensitive indicator of such disease. The blood urea nitrogen (BUN) test, a routine but critical clinical laboratory assay used to evaluate renal function, quantifies serum urea that is produced in the liver as an endpoint of protein degradation. Studies are proposed that will lead to the development of a fluorescence-based biosensor capable of quantifying urea in a sample in real time. The sensor will employ the UreR protein encoded by the urease operon of the uropathogenic bacterial species Proteus mirabilis. UreR, to date, is the only documented urea-binding protein. When UreR binds urea, the protein undergoes a conformational change that is detectable using fluorescence technology. Three approaches to biosensor design will include fluorescent labeling of genetically modified UreR, fluorescence resonance energy transfer of UreR-Yellow-fluorescent-protein fusions, and an anisotropy approach using the binding of its target DNA by UreR. The proposed UreR biosensor will directly measure urea in real time without the necessity of hydrolyzing urea with urease, which is required in current sensor technology. Primary applications may include routine blood urea nitrogen determination and monitoring of blood urea nitrogen directly in the blood of patients undergoing kidney dialysis. In addition, the biosensor will be useful in basic science investigations including urea transport in the liver and kidney, the role of urea hydrolysis in urolithiasis, and urea metabolism in the gastric mucosa infected by urease-positive Helicobacter pylori. This biosensor also offers the prospect of imaging urea concentration and fluxes in tissue using fluorescence and confocal microscopy. Our experimental approaches are organized into three Specific Aims: 1) To define the urea binding domain of the UreR protein 2) to develop a urea sensor using UreR; and 3) to construct a prototype UreR-based urea sensor and validate it in specific applications.
