Generation of Membrane Protein Production Strains(RMI)

描述(由申请人提供)：膜蛋白物理和结构表征的主要障碍之一是它们在标准方法中的表达通常很差。该项目试图通过创造更有效的膜蛋白产生菌来缓解这一瓶颈。我们计划采用遗传方法，使用我们开发的膜蛋白表达的强大选择。通过识别和表征表达突变体，我们还计划更多地了解表达的主要障碍。我们的长期目标是将在这里学到的经验应用于其他生物，进一步扩大可以高水平表达的膜蛋白的范围。具体的目标是：目标I.寻找(当过度表达或突变时)可以改善低表达的膜蛋白的产生和/或将表达从包涵体重定向到膜的基因。目的II.确定基因/突变。目的III.对AIM II中确定的基因进行定向突变，以寻找更有效的表达突变体。结合最有效的突变体构建了一套膜蛋白表达菌株。目的IV.研究表达突变体对膜蛋白生物发生过程的影响。与公共卫生相关：膜蛋白在从囊性纤维化到癌症的各种疾病中发挥着重要作用，是绝大多数药物的靶标。我们无法获得足够数量的膜蛋白用于详细研究，这阻碍了我们对这些疾病的了解和开发新药的能力。这个项目是通过将细菌转化为膜蛋白生产工厂来减少这一问题的努力。

DESCRIPTION (provided by applicant): One of the primary barriers to the physical and structural characterization of membrane proteins is their often poor expression with standard methods. This project seeks to alleviate this bottleneck by creating E. coli strains that are more effective membrane protein producers. We plan to take a genetic approach, using a powerful selection for membrane protein expression that we have developed. By identifying and characterizing expression mutants we also plan learn more about the major barriers to expression. Our longer term goals are to apply the lessons learned here to other organisms, further broadening the range of membrane proteins that can be expressed at high levels. The specific aims are: Aim I. Find genes that (when over-expressed or mutated) can improve the production of poorly expressed membrane proteins and/or that redirect expression from inclusion bodies into membranes. Aim II. Identify the genes/mutations. Aim III. Employ targeted mutagenesis of the genes identified in Aim II to find even more effective expression mutants. Combine the most effective mutants to build a set of membrane protein expression strains. Aim IV. Characterize the effects of the expression mutants on the steps of membrane protein biogenesis. Relevance to Public Health: Membrane proteins play significant roles in diseases ranging from cystic fibrosis to cancer, and are the targets of the vast majority of drugs. Our understanding of these diseases and our ability to develop new drugs has been hampered by our inability to obtain sufficient quantities of many membrane proteins for detailed study. This project is an effort to reduce this problem by converting bacteria into membrane protein production factories.