The microbial eukaryote Pichia pastoris will be developed as an expression system to generate large quantities of recombinant proteins that are completely and uniformly 13C and 15N-labeled (i.e. isotopically labeled). The proteins produced in this system win be ideal for high resolution, multi-dimensional NMR studies. NMR data generated using proteins produced by this technology can be used for dynamic protein structural analysis and the elucidation of protein-ligand interactions, both which serve as a basis for rational drug design by providing a high resolution structure of proteins and interaction with their inhibitors, modulators, and activators. Pichia is able to grow on simple, defined media that can include as its only carbon source, methanol, and as its only nitrogen source, ammonium sulfate or ammonium chloride. Methanol can be used not only as a carbon source but it also functions as an inducer of an expression system capable of producing large amounts of recombinant protein. The optimal conditions will be determined to achieve complete and uniform 13C and 15N labeling of expressed recombinant protein when the sole source of carbon and nitrogen is methanol (13CH3OH) and ammonium sulfate or ammonium chloride ([15NH4]2SO4 or 15NH4Cl), respectively.
