Membrane Protein Production Using the Yeast SPP System

描述(由申请人提供)：膜蛋白在动态的细胞过程中发挥关键作用，这对维持动态平衡和人类健康是必不可少的。确定这些蛋白质的结构是对了解它们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的蛋白质的困难的阻碍。从细菌表达系统获得的真核蛋白质的未折叠特性被归因于缺乏翻译后修饰，缺乏影响折叠的伴侣或其他真核过程，以及蛋白质在自然界中内在展开的可能性。第一代真核蛋白表达系统已经被开发出来，它具有提高正确折叠蛋白的表达和回收的潜力。这个系统被称为酵母SPP系统，它使用酿酒酵母和一种名为MazF的细菌毒素来给予生长停滞状态，允许重组蛋白继续表达，而不会出现通常因过度表达而导致的毒性，从而增加目标蛋白的产量，同时减少酵母蛋白的本底。长期目标是建立酵母SPP系统，以生产用于结构研究的具有生物重要性的膜蛋白。填补结构-功能研究的这一空白是开发与膜蛋白相关疾病的新治疗方法的重要关键。这项提议将建立概念证明，即这种新的方法能够通过实现三个特定目标来实现正确折叠的真核蛋白质的稳健生产。目的1以人嗜酸性粒细胞趋化蛋白为模型靶蛋白，利用酿酒酵母和MazF毒素对第一代酵母SPP系统进行优化。将酵母SPP产生的嗜酸性粒细胞趋化蛋白的核磁共振数据与传统方法进行比较，将确定这项技术的实用性。目标2将进一步调整SPP系统，以表达和纯化选定的酵母和人膜蛋白，其中结构信息可用。异核单量子相干(HSQC)和主干共振归属分析将验证酵母SPP系统产生的蛋白质。目标3将进一步扩展这些研究，以生产参与葡萄糖运输的具有生物学意义的膜蛋白。这些研究符合美国国立卫生研究院结构生物学路线图的使命，提供了一项重大的技术进步，将推进膜蛋白结构的研究。 与公共卫生相关：了解细胞表面蛋白质的结构，称为膜蛋白，可以为了解它们在健康和疾病中的作用提供重要的见解。然而，以其自然形式生产和纯化这些蛋白质是困难的。该项目将开发使用酵母生产人膜蛋白的新技术，这些蛋白可以被研究，以促进我们对它们如何发挥作用的了解，从而产生治疗疾病的新方法。

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.