Functional expression of CFTR and other multidomain proteins

描述(申请人提供)：蛋白质的高分辨率结构测定是理解细胞功能的分子机制的关键。遗传病中最常见的是改变蛋白质结构/功能的突变。获得野生型和突变型蛋白质的高分辨率结构的一个主要障碍是难以在结构研究所需的水平上在同源或异源系统中表达感兴趣的蛋白质。目前表达可溶性蛋白的方法，特别是膜蛋白，既依赖于合理的设计，也依赖于偶然性。过度表达蛋白质的能力可能是个谜，也可能是结构确定的主要障碍。囊性纤维化跨膜电导调节因子(CFTR)是一种关键蛋白质，其结构分析因无法表达大量蛋白质而受到限制。这个项目将测试一个新的假说，它直接解决了蛋白质低表达的原因。最重要的是，cftr是检验这一假说的理想蛋白质，因为假说预测cftr很难在高水平表达。因此，CFTR的高水平表达和降低CFTR表达的对照组将是对这一假说的关键检验。此外，CFTR是至关重要的，其内部的突变是导致一种主要的世界性疾病的原因。总体而言，如果成功，该项目的结果将对包括囊性纤维化研究在内的生物医学领域产生很大影响。与公共健康相关：基于新的和已知蛋白质的高分辨率结构的合理药物设计需要蛋白质的高水平表达系统。功能蛋白的高水平表达是分析大多数膜蛋白的限制因素，包括囊性纤维化中的缺陷蛋白CFTR。这个项目将检验一个新的假说，如果这个假说正确的话，它将为成功表达多结构域蛋白提供一种合理的方法，并使用CFTR作为测试案例。

DESCRIPTION (provided by applicant): High-resolution structure determination of proteins is critical in understanding the molecular mechanisms of cellular function. The greatest occurrences within genetic diseases are mutations that alter the structure/function of proteins. A major obstacle for obtaining the high-resolution structure of wild type and mutant proteins is the difficulty in expressing proteins of interest in either a homologous or heterologous system at levels needed for structural studies. The current methods for expressing soluble, and especially membrane, proteins rely on fortuity as much as rational design. The inability to over express proteins can be enigmatic and can be a major block for structure determination. The cystic fibrosis transmembrane conductance regulator (CFTR) is a prime example of a critical protein whose structural analysis has been restricted by the inability to express large amounts of the protein. This project will test a novel hypothesis, which directly addresses the cause for low expression of proteins. CFTR is an ideal protein to use to test this hypothesis most importantly because CFTR is predicted by the hypothesis to be difficult to express at high levels. Thus, expression of CFTR at high levels and controls, which reduced expression of CFTR, would be critical tests for the hypothesis. In addition, CFTR is critical and mutations within it are responsible for a major worldwide disease. Overall, if successful, the results of this project will have a high impact on the biomedical field including research on cystic fibrosis. PUBLIC HEALTH RELEVANCE: Rational drug design based on the high resolution structure of novel and known proteins requires systems for high level expression of the proteins. The high level expression of functional protein is a limiting factor for analysis of most membrane proteins, including the protein defective in cystic fibrosis, CFTR. This project will test a novel hypothesis, which if correct will provide a rational approach for successful expression of multidomain proteins with CFTR being used as the test case.