HIGH THROUGHPUT XRAY STRUCTURES OF MEMBRANE PROTEINS

据估计，三分之一的脊椎动物蛋白质是膜结合的。不幸的是，由于膜蛋白的疏水性，膜蛋白通常是高度不溶的，而且出了名的难以纯化和结晶。如果“结构基因组学计划”要在合理的时间尺度上取得成功，膜蛋白的X射线结构测定必须大大简化。因此，我们的目标是开发一套完整的研究工具，旨在以普遍适用的方式快速克隆、表达、纯化、结晶和确定膜蛋白的X射线结构。具体地说，我们建议证明，人拓扑异构酶I(Topo I)可以用于以选择的方向将PCR产物快速亚克隆到任何商业表达载体中。我们还打算证明，各种膜蛋白可以被制成嵌合蛋白，这些嵌合蛋白易于表达、纯化和与准固体阳离子脂双层共结晶。这些努力的成功将促进高通量X射线结构测定，不仅是膜蛋白，而且是任何可能由于疏水性质而抵抗结晶的蛋白质。拟议的商业应用：如果成功，这一第一阶段项目将成为膜蛋白X射线结构测定的一般和综合方法的基础。此外，由于工业生物技术的主要部分涉及涉及膜受体蛋白的信号通路，因此如果我们的方法成功，将对Emerald BioStructures提供的结晶学研究服务产生非常大的需求。因此，这项研究不仅有助于膜蛋白的高通量结构测定，而且有助于催生一个新的大分子结晶学服务产业。

It is estimated that one third of all vertebrate proteins are membrane bound. Unfortunately, by virtue of their hydrophobic nature, membrane proteins are often highly insoluble and are notoriously difficult to purify and crystallize. If the "Structural Genomics Initiative" is to succeed on a reasonable time scale, the X-ray structure determination of membrane proteins must be greatly simplified. Therefore, our goal is to develop an integrated set of research tools that are designed to enable the rapid cloning, expression, purification, crystallization and X-ray structure determination of membrane proteins in a generally applicable manner. Specifically, we propose to demonstrate that human topoisomerase I (topo I) can be used in to rapidly sub-clone PCR products into any commercial expression vector, in an orientation of choice. We also intend to demonstrate that a variety of membrane proteins can be made as chimeric proteins that are simple to express, purify, and co-crystallize with quasi-solid cationic lipid bilayers. Success in these endeavors will facilitate the high-throughput X-ray structure determination of not just membrane proteins, but any protein that may resist crystallization due to its hydrophobic nature. PROPOSED COMMERCIAL APPLICATION: If successful, this Phase I project would form the basis for a general and integrated approach towards the X-ray structure determination of membrane proteins. Moreover, since a major portion of industrial biotechnology is concerned with signaling pathways involving membrane receptor proteins, success in our approach would create a very large demand for the crystallography research services offered by Emerald BioStructures. Hence, the proposed research will not only facilitate the high-throughput structure determination of membrane proteins, but will help to spawn a new service industry in macromolecular crystallography.