VECTORIAL CHARACTERIZATION OF NATIVE CFTR

自从CFTR基因被发现以来已经过去了将近五年， 测序 然而，调查人员无法提供足够的证据， 天然CFTR的数量，以便其功能可以精确定义 并在体外进行了广泛的研究。 因此，我们的分子 对CFTR（一种完整的膜蛋白）的理解还远远落后 膜生物学领域的建立。 为了解决这个问题 严重的问题，这个项目将首先集中在几个小说 生产大量天然CFTR，然后利用 获得的制剂，以确定其在纯 脂质体膜系统。 具体目标有五个方面，即： 1. 在杆状病毒/昆虫系统中共表达CFTR，具有一个或多个 分子伴侣，以促进大量生产 正确折叠的分子 2. 使用一种或多种快速繁殖的寄生虫 生物，我们已经证明含有CFTR样蛋白，作为一种 替代的真核过表达系统。 3. 利用这些表达系统， 最近在E.大肠杆菌，以功能形式， 另外两种膜蛋白，完整的ATP合酶和阴离子 载体 4. 加入获得的那些纯CFTR制剂，其表现出 重要的二级结构，进入脂质体囊泡，并评估其 在ATP存在下催化氯化物渗透的能力， Mg++。 5. 确定磷酸化、ATP水解和 CFTR需要逆流，以促进氯化物 渗透性，以及这些或其他条件是否有利于通道或 类似泵的功能。 这项工作对于我们理解 载体的性质，因此分子功能和功能障碍的 CFTR分子，并且应该对SCOR团队的其他成员非常宝贵 帮助解释体内数据。

Almost five years have elapsed since the CFTR gene was identified and sequenced. Yet, investigators have been unable to produce sufficient amounts of native CFTR so that its function(s) can be precisely defined and extensively studied in vitro. For this reason, our molecular understanding of CFTR, an integral membrane protein, lies far behind established areas of membrane biology. To help resolve this rather serious problem, this project will focus first on several novel approaches to produce large amounts of native CFTR and then utilize the preparation(s) obtained to define its ATP-dependent function(s) in a pure liposomal membrane system. SPECIFIC AIMS ARE FIVE FOLD AND WILL BE TO: 1. Coexpress CFTR in a baculoviral/insect system with one or more molecular chaperons in order to facilitate the abundant production of properly folded molecules. 2. Employ the use of one or more rapidly proliferating parasitic organisms, which we have shown to contain a CFTR-like protein, as an alternative eukaryotic overexpression system. 3. Utilization those expression systems which have resulted very recently in the large scale production in E. coli, in functional form, of two other membrane proteins, the complete ATP synthase, and an anion carrier. 4. Incorporate those pure CFTR preparations obtained, which exhibit significant secondary structure, into liposomal vesicles and assess their capacity to catalyze chloride permeability in the presence of ATP and Mg++. 5. Establish the extent to which phosphorylation, ATP hydrolysis, and counterion flow are required for CFTR to facilitate chloride permeability, and whether these or other conditions favor a channel or pump-like function. This work is both necessary and fundamental to our understanding of the vectorial nature and hence the molecular function and dysfunction of the CFTR molecule, and should be invaluable to other members of the SCOR team in helping interpret data derived in vivo.