Membrane Protein Production Using the Yeast SPP System

描述（由申请人提供）：膜蛋白在动态细胞过程中起关键作用，对维持体内平衡和人体健康至关重要。确定这些蛋白质的结构是对理解其功能的经典遗传和生化方法的补充，但结构分析一直受到表达和纯化正确折叠蛋白质的困难的阻碍。从细菌表达系统中获得的真核蛋白的未折叠性质归因于缺乏翻译后修饰，缺乏伴侣或其他影响折叠的真核过程，以及蛋白质在本质上是未折叠的可能性。第一代真核蛋白表达系统已经开发出来，有可能提高正确折叠蛋白的表达和恢复。这个系统被称为酵母SPP系统，它使用酿酒酵母和一种叫做MazF的细菌毒素来赋予一种生长停滞状态，这种状态允许重组蛋白继续表达，而不会产生因过度表达而经常引起的毒性，从而增加目标蛋白的产量，同时减少酵母蛋白的背景。长期目标是建立酵母SPP系统，以生产生物学上重要的膜蛋白用于结构研究。填补这一结构-功能研究的空白是开发与膜蛋白相关疾病的新治疗方法的重要关键。该提案将建立概念证明，这种新方法能够通过实现三个特定目标来实现正确折叠真核蛋白的稳健生产。目的1将利用酿酒酵母和MazF毒素优化第一代酵母SPP系统，生产人eotaxin作为模型靶蛋白。将酵母SPP与传统方法制备的eotaxin的NMR数据进行比较，将确定该技术的实用性。Aim 2将进一步调整SPP系统用于表达和纯化选定的酵母和人膜蛋白，其中结构信息是可用的。杂核单量子相干（HSQC）和主链共振分配分析将验证酵母SPP系统产生的蛋白质。Aim 3将进一步扩展这些研究，以产生参与葡萄糖运输的生物重要膜蛋白。这些研究通过提供将推进膜蛋白结构研究的重大技术进步，符合NIH结构生物学路线图的使命。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Understanding the structure of proteins found on the surface of cells, called membrane proteins, can provide important insight into their role in health and disease. However, it is difficult to produce and purify these proteins in their natural form. This project will develop new technology using yeast to produce human membrane proteins that can be studied to advance our knowledge of how they function, thereby generating new approaches to treat disease.