Protein and enzyme engineering and drug discovery would both benefit from a general method of applying the power of genetic selection. To this end, we have developed chemical complementation. This yeast-based three-component system (a derivative of the yeast two-hybrid system) links the survival of cells to the presence of a specific small molecule. This system comprises a human nuclear receptor protein, its coactivator protein, and a small molecule ligand, where the nuclear receptor and coactivator associate and activate transcription only in the presence of the small molecule ligand. Our chemical complementation system is highly sensitive (producing growth at 10 nM ligand concentration), has zero background (no growth without ligand) and high dynamic range (growth density and time equivalent to Gal4 induced growth). To develop a system with the versatility of both positive and negative selection, we created a yeast strain with the URA3 gene and developed negative chemical complementation. In this system, the presence of the ligand and receptor causes cell death in media containing 5'-fluoroorotic acid. Both chemical complementation and negative chemical complementation can greatly benefit protein engineering, where chemical complementation offers a general method of engineering receptors that activate transcription in response to arbitrary small molecules. Engineered proteins will have broad uses as biosensors, in metabolic engineering, and gene therapy. This method also promises the first general method to engineer enzymes through genetic selection and screen otherwise intractable enzymes. For drug discovery, positive and negative chemical complementation assays are described for discovering nuclear receptor agonists and antagonists. Nuclear receptors are currently targets for ~10% of commonly prescribed drugs and potentially many more. Furthermore, drug discovery assays may be extended to enzyme targets by engineering receptors that activate transcription in response to the small molecule product of the enzyme-catalyzed reaction. In this type of a system, chemical complementation is used to engineer the receptor, as well as provide selective pressure on the cell to produce ample amounts of the small molecule product, providing an advantage for metabolic engineering.

Extended Abstract Status: Not Uploaded