Eukaryotic System to Express Labeled Proteins for NMR

The microbial eukaryote Pichia pastoris will be developed as an expression system to generatelarge 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-dimensi-onal NMR studies. NMR data generated using proteins produced by this technology can be used fordynamic protein structural analysis and the elucidation of protein-ligand interactions, both which serveas a basis for rational drug design by providing a high resolution structure of proteins and interactionwith their inhibitors, modulators, and activators. Pichia is able to grow on simple, defined media thatcan include as its only carbon source, methanol, and as its only nitrogen source, ammonium sulfate orammonium chloride. Methanol can be used not only as a carbon source but it also functions as aninducer of an expression system capable of producing large amounts of recombinant protein. Theoptimal conditions will be determined to achieve complete and uniform 13C and 15N labeling ofexpressed recombinant protein when the sole source of carbon and nitrogen is methanol (13CH3OH) andammonium sulfate or ammonium chloride ([15NH4]2SO4 or 15NH4Cl), respectively.