Membrane Protein Production using Retroviral Vehicles

描述（由申请人提供）：尽管它们很重要，但多次跨越膜的蛋白质对结构分析（如X射线晶体学）提出了一系列独特的挑战。这些蛋白质的大部分是疏水性的，蛋白质在拓扑上是复杂的，并且从它们的脂质双层去除导致它们的天然结构的损失。由于其复杂的折叠和运输，许多膜蛋白，特别是真核生物来源的膜蛋白，难以表达。表达限制有时可以克服，但膜蛋白仍然必须从细胞中纯化出来，这一过程受到裂解物中蛋白质异质性、不需要的膜蛋白和可用去污剂的限制。如果没有足够数量的结构完整的膜蛋白开始，结构分析的许多下游步骤不能优化，甚至启动。虽然目前还不确定常规获得膜蛋白晶体结构需要取得哪些进展，但很明显，传统的材料和方法是不够的，特别是对于真核细胞膜蛋白。该提案的目的是开发一种能够产生多毫克数量的结构完整的、均质的膜蛋白的系统，所述膜蛋白可用于结构分析。

DESCRIPTION (provided by applicant): Despite their importance, proteins that span the membrane multiple times present a unique set of challenges for structural analyses such as x-ray crystallography. Large portions of these proteins are hydrophobic, the proteins are topologically complex, and removal from their lipid bilayer results in loss of their native structure. Because of their complex folding and trafficking, many membrane proteins, especially those of eukaryotic origin, are difficult to express. Expression limitations can sometimes be overcome, but the membrane proteins must still be purified away from the cell, a process that is limited by protein heterogeneity in lysates, unwanted membrane proteins, and available detergents. Without sufficient quantities of structurally intact membrane protein to start with, many of the downstream steps of structural analysis cannot be optimized or even initiated. Although it is not certain what advances need to be made to routinely obtain membrane protein crystal structures, it is clear that traditional materials and methods are not sufficient, especially for eukaryotic membrane proteins. The purpose of this proposal is to develop a system capable of producing multi-milligram quantities of structurally intact, homogeneous membrane protein that can be used for structural analysis.