Membrane proteins play a critical role in dynamic cellular processes that are essential to maintain homeostasis and human health. Determining the structure of these proteins complements classical genetic and biochemical approaches to understanding their function, yet structural analysis has been hampered by difficulties in expressing and purifying proteins that are properly folded. The unfolded nature of eukaryotic proteins obtained from bacterial expression systems has been attributed to lack of post-translational modifications, the absence of chaperones or other eukaryotic processes that influence folding, and the possibility that the protein is intrinsically unfolded in nature. A first generation expression system for eukaryotic protein expression has been developed that has the potential to improve the expression and recovery of correctly folded proteins. This system, called the yeast SPP system, uses Saccharomyces cerevisiae and a bacterial toxin called MazF to impart a state of growth arrest that allows continued expression of recombinant protein without the toxicity that is frequently caused by overexpression, resulting in an increased yield of the target protein coupled with reduced background of yeast proteins. The long-term goal is to establish the yeast SPP system to produce biologically-important membrane proteins for structural studies. Filling this gap in structure-function studies represents an important key to developing new therapeutic approaches for diseases linked to membrane proteins. This proposal will establish proof-of-concept that this novel approach is capable of achieving robust production of properly folded eukaryotic proteins through the accomplishment of three specific aims. Aim 1 will optimize the first generation yeast SPP system using Saccharomyces cerevisiae and the MazF toxin by producing human eotaxin as a model target protein. Comparison with NMR data from eotaxin produced by yeast SPP vs. conventional methods will establish the utility of this technology. Aim 2 will further adapt the SPP system for the expression and purification of selected yeast and human membrane proteins where structural information is available. Heteronuclear Single Quantum Coherence (HSQC) and backbone resonance assignment analysis will validate proteins produced by the yeast SPP system. Aim 3 will extend these studies further to produce biologically-important membrane proteins involved in glucose transport. These studies fit with the mission of the NIH Structural Biology Roadmap by providing a significant advance in technology that will advance the study of membrane protein structure.