FUNCTIONAL ANALYSIS OF CFTR

作为其长期目标，该项目旨在了解 囊性纤维化跨膜传导功能 调节器），其缺陷导致囊性纤维化的蛋白质。 的 实现这一目标所需的具体目标包括：（1）提高 与CFTR肽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。 等 然后将纯化的（和可能变性的）材料用于获得（2） 一组单克隆抗体， 特定的抗原决定簇。 在筛选这些第二代的时候 抗体，应该可以识别那些高 亲和力和低亲和力的那些。(3)低亲和力抗体 将用于第二轮免疫纯化， 预防措施和温和的条件，旨在促进保留 生物活性(4)在平行工作中，CFTR也将通过以下方法纯化： 生物化学技术，从人类细胞系（T84）开始， 推测具有高水平的CFTR，或具有过表达系统 使用全长cDNA设计。 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）具有核苷酸结合折叠。 如果早期的工作（以上） 证明困难，这提供了另一个机会，以获得抗原， 单克隆抗体组的产生。 有了纯化的CFTR，两个基本问题将是 直接处理。(1)更具挑战性的实验将确定 增强或抑制ATP水解活性的内源性物质 孤立的CFTR 这是可能的（但不确定），CFTR运输 分子进入或离开膜结合区室（细胞，一个小分子）。 细胞器等），这种基质是 CFTR。 从其他系统论证，应该可以识别出这一点 关键分子（和其他效应物）通过刺激CFTR ATP酶 活动 这将立即为有效的 用于治疗囊性纤维化的药理学。(2)的第二集合中 实验中，高亲和力的单克隆抗体将用于 将CFTR固定到亲和柱上。 这将允许表征 与CFTR本身相互作用的蛋白质。 这些蛋白质将成为 控制上皮细胞的调节网络的候选成员 功能 总之，这些实验有可能揭示两者 靶向CFTR小分子和大分子。 这种资料应 构成了关于膜功能是如何 在正常和疾病状态下都受到调节。

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.