Functional expression of CFTR and other multidomain proteins

CFTR 和其他多域蛋白的功能表达

• 批准号: 7904914
• 负责人: Neil A Bradbury
• 金额: $ 15.4万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904914
• 关键词: Address; Affect; Cell membrane; Cell physiology; Cells; Code; Codon Nucleotides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease; Drug Design; Effectiveness; Embryo; Factor Analysis; Genes; Hereditary Disease; Human; Membrane; Membrane Proteins; Messenger RNA; Methods; Molecular; Mutation; Physiologic pulse; Plasmids; Proteins; Relative (related person); Research; Resolution; Structure; System; Tertiary Protein Structure; Testing; Time; Translations; Water; base; design; interest; kidney cell; mutant; novel; patch clamp; protein expression; protein folding; protein structure function; public health relevance; research study

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.

描述（由申请人提供）：蛋白质的高分辨率结构测定对于理解细胞功能的分子机制至关重要。遗传疾病中最常见的是改变蛋白质结构/功能的突变。获得野生型和突变蛋白的高分辨率结构的主要障碍是难以在同源或异源系统中以结构研究所需的水平表达感兴趣的蛋白。目前表达可溶性，尤其是膜蛋白的方法依赖于偶然性和合理设计。不能过度表达蛋白质可能是神秘的，并且可能是结构确定的主要障碍。囊性纤维化跨膜传导调节因子（CFTR）是一个重要的蛋白质，其结构分析受到无法表达大量蛋白质的限制。该项目将测试一种新的假设，该假设直接解决了蛋白质低表达的原因。CFTR是一种理想的蛋白质，用于测试这一假设，最重要的是，因为CFTR是预测的假设是难以表达在高水平。因此，高水平的CFTR表达和降低CFTR表达的对照将是该假设的关键检验。此外，CFTR是至关重要的，其中的突变是导致全球重大疾病的原因。总的来说，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重大影响。公共卫生关系：基于新的和已知的蛋白质的高分辨率结构的合理药物设计需要用于蛋白质的高水平表达的系统。功能蛋白的高水平表达是大多数膜蛋白分析的限制因素，包括囊性纤维化缺陷蛋白CFTR。该项目将测试一个新的假设，如果该假设正确，将以CFTR作为测试案例，为多结构域蛋白的成功表达提供一种合理的方法。