Single Protein Production in Yeast Cells

描述（由申请人提供）：以快速和廉价的方式解决蛋白质三维结构的能力对结构基因组学的使命至关重要。蛋白质结构为基础科学和临床知识库增加了有价值的信息。在临床方面，这些结构可用于阐明重要疾病相关蛋白的功能，用于识别影响人类健康的关键信号蛋白的抑制剂或增强剂，或用于识别药物靶点。目前，细菌表达系统最常用于核磁共振（NMR）和x射线晶体学的结构研究。我们最近在大肠杆菌中发现了一种ACA序列特异性核糖核酸内切酶，称为MazF。我们的初步研究表明，具有MazF活性的细胞促进了来自无ACA序列的mrna的高水平蛋白质的表达。相反，由于宿主细胞mrna中ACA序列的丰富，只有微量的背景细胞蛋白合成发生。这项RO1提案的目的是基于这样的假设：将MazF核糖核酸内切酶的独特特性应用于酵母细胞表达系统将提高一系列正确折叠的真核蛋白的有效表达和恢复。将利用MazF表达的独特特性开发酵母单蛋白生产（SPP）系统。这些属性应该有助于核磁共振和x射线结构研究，而无需实施蛋白质纯化步骤，并且可能允许使用核磁共振直接可视化活细胞中的蛋白质结构。目的1提出了在酿酒酵母中开发基于mazf的SPP系统的实验。目标2将在酿酒酵母中开发的技术应用于克卢维酵母的乳酸中，以努力提高产品产量。在Aim 3中，将研究MazF在酵母细胞上表达的生理后果，以便使用SPP系统优化表达。最后，在Aim 4中，SPP系统将应用于表达细菌时展开的真核蛋白子集。在异核单量子相干分析中显示正确折叠的蛋白质将被核磁共振用于结构测定。

DESCRIPTION (provided by applicant): The ability to solve the three dimensional structure of a protein in a rapid and inexpensive manner is instrumental to the Structural Genomics mission. Protein structures add valuable information to the basic science and clinical knowledge base. On the clinical side, these structures can be instrumental for illuminating the function of important disease related proteins, for identifying inhibitors or enhancers of key signaling proteins that influence human health or for identification of drug targets. Currently, bacterial expression systems are most commonly enlisted for structure studies using nuclear magnetic resonance (NMR) and X-ray crystallography. We have recently discovered an ACA sequence-specific endoribonuclease in E. coli called MazF. Our preliminary studies demonstrate that cells possessing MazF activity facilitate the expression of high levels of protein derived from mRNAs engineered without ACA sequences. In contrast, because of the abundance of ACA sequences in host cell mRNAs, only trace amounts of background cellular protein synthesis occurs. The aims of this RO1 proposal are based on the hypothesis that application of the distinctive properties of MazF endoribonuclease to yeast cell expression systems will enhance the efficient expression and recovery of an array of correctly folded eukaryotic proteins. The unique properties of MazF expression will be exploited for development of a yeast single protein production (SPP) system. These attributes should facilitate NMR and X-ray structural studies without having to implement a protein purification step and will likely allow for direct visualization of protein structures in living cells using NMR. Aim 1 proposes experiments for the development of a MazF-based SPP system in yeast Saccharomyces cerevisiae. Aim 2 applies the technology developed in yeast S. cerevisiae to Kluyveromyce's lactis in an effort to amplify product yields. In Aim 3, the physiological consequences of MazF expression on yeast cells will be studied in order to optimize expression using the SPP system. Finally, in Aim 4 the SPP system will be applied to a subset of eukaryotic proteins that are unfolded when expressed bacteria. Proteins that display proper folding upon Heteronuclear Single Quantum Coherence analysis will then be subjected to NMR for structural determination.