FUNCTIONAL ANALYSIS OF CFTR

作为其长期目标，该项目试图了解 囊性纤维化跨膜电导的作用 调节因子)，这种蛋白质的缺陷会导致囊性纤维化。这个 实现这一目标所需的具体目标包括：(1)提高 对CFTR多肽有反应的多克隆或单克隆抗体；这些 可以作为免疫吸附剂来纯化完整的分子。是这样的 然后将使用提纯的(和可能变性的)材料来获得(2) 一组对更广泛的范围起反应的单抗 特定的表位。在筛选这些第二代人的过程中 抗体，应该可以识别出那些高水平的抗体 亲和力和低亲和力。(3)低亲和力抗体 将用于第二轮免疫纯化，具有特定的 旨在促进留存的预防措施和温和条件 生物活性。(4)在并行工作中，CFTR也将得到净化 生化技术，从人类细胞系(T84)开始 推测有高水平的CFTR，或具有过度表达系统 设计使用全长cdna。这些生化步骤将包括 使用与蛋白质相互作用的三嗪染料偶联树脂(例如 Cftr)有核苷酸结合褶皱的。之前的工作是否应该(上) 事实证明很难，这为获得抗原提供了另一个机会 单抗小组的制作。 有了纯化的CFTR，将有两个基本问题 直接寻址。(1)更具挑战性的实验将确定 增强或抑制ATP水解酶活性的内源物质 孤立的CFTR。CFTR很可能(但不确定)会输送一种 分子进入或离开膜结合的隔室(细胞，小分子 细胞器等)，而这种底物是运行的关键 CFTR。从其他系统的观点来看，应该可以确定这一点 CFTRATPase激活的关键分子(和其他效应分子) 活动。这将立即为有效的 治疗囊性纤维化的药理学。(2)在第二套 实验中，高亲和力的单抗将被用于 将CFTR粘贴到亲和柱上。这将允许对 与cftr本身相互作用的蛋白质。这样的蛋白质会变成 候选人是控制上皮细胞的监管网络的成员 功能。总而言之，这些实验有可能揭示两者 针对cftr的小分子和大分子。这样的信息应该 构成了关于膜功能的核心知识 在正常和疾病状态下都受到监管。

As its long term objective, this project seeks to understand the function of CFTR (Cystic Fibrosis Transmembrane conductance Regulator), the protein whose defect causes cystic fibrosis. The specific aims required to achieve this objective include (1) the raising of polyclonal or monoclonal antibodies reactive to CFTR peptides; these can serve as immunoadsorbents to purify the intact molecule. Such purified (and likely denatured) material will then be used to obtain (2) a panel of monoclonal antibodies reactive against a very much wider range of specific epitopes. During the screening of these second generation antibodies, it should be possible to identify those that are high affinity and those that are low affinity. (3) Low affinity antibodies will be used in a second round of immunopurification, with specific precautions and mild conditions designed to promote retention of biological activity. (4) In parallel work, CFTR will also be purified by biochemical techniques, beginning with either a human cell line (T84) presumed to have high levels of CFTR, or with an overexpression system designed using full length cDNA. These biochemical steps will include use of triazine dye-coupled resins that interact with proteins (e.g. CFTR) having nucleotidebinding folds. Should the earlier work (above) prove difficult, this provides another opportunity to obtain antigens for production of a monoclonal antibody panel. With purified CFTR available, two fundamental issues will be addressed directly. (1) The more challenging experiments will identify endogenous materials that enhance or inhibit the ATP hydrolytic activity of isolated CFTR. It is likely (but not certain) that CFTR transports a molecule into or out of a membrane-bound compartment (the cell, a minor organelle, etc.), and that this substrate is key to the operation of CFTR. Arguing from other systems, it should be possible to identify this key molecule (and other effectors) by a stimulation of CFTR ATPase activity. This will immediately set the stage for an effective pharmacology for treatment of cystic fibrosis. (2) In the second set of experiments, the high affinity monoclonal antibodies will be used to affix CFTR to an affinity column. This will allow a characterization of proteins that interact with CFTR itself. Such proteins will become candidates members of a regulatory network that controls epithelial cell function. Together, these experiments have potential for revealing both small and large molecular targeted to CFTR. Such information should constitute the central core of knowledge about how membrane function is regulated in both normal and disease states.