DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尽管迫切需要更好的方法来表达和研究完整的膜蛋白，但几乎没有系统的研究来确定导致未能恢复高水平活性蛋白的特征。Grisshammer和Tate(1995)提出，膜蛋白过度表达系统的进展需要了解膜蛋白如何折叠，以及哪些细胞蛋白参与了自然环境中的成功折叠。我们的假设是，只有对类似膜蛋白家族的膜蛋白表达的分子和细胞限制进行系统检查，才能改善过度表达。这是该子项目表达部分的重点，该部分有两个目标：确定两个主要表达系统中膜蛋白表达的限制，并根据需要重新设计蛋白质和表达系统以改善表达。 即使有足够的表达水平，膜蛋白的研究也受到提取和有时折叠蛋白质的困难的限制。因此，我们还将建立一种策略，以选择最适合纯化、增溶和稳定天然结构的洗涤剂和脂类。这将包括研究表面活性剂体系本身的性质，特别是相行为，以及它们在膜蛋白存在时的行为。除了指导重新折叠战略外，这些信息本身也将是有用的。我们的结果将增加对膜蛋白插入、折叠以及与洗涤剂和脂类相互作用机制的了解，并将促进大量活性完整膜蛋白的生产。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite the clear and urgent need for better methods to express and study integral membrane proteins, there have been few if any systematic studies to identify the features that lead to the failures to recover high levels of active protein. Grisshammer and Tate (1995) suggested that advances in overexpression systems of membrane proteins required an understanding of how membrane proteins fold and of which cellular proteins are involved in successful folding in the native environment. Our hypothesis has been that only a systematic examination of the molecular and cellular limitations to membrane protein expression for similar families of membrane proteins will lead to improvements in overexpression. This is the focus of the expression component of this subproject, which has two goals: identify the limitations to membrane protein expression in two major expression systems and re-engineer both protein and the expression systems as needed to improve expression. Even with adequate expression levels, the study of membrane proteins is limited by difficulties in extracting and sometimes refolding the proteins. We will therefore also establish a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure. This will include studies of the properties, particularly phase behavior, of the surfactant systems themselves, as well as of their behavior in the presence of membrane proteins. In addition to guiding refolding strategies, this information will be useful in its own right. Our results will provide increased knowledge of the mechanism of membrane protein insertion, folding, and interactions with detergents and lipids, and will facilitate production of significant quantities of active integral membrane proteins.