DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。尽管迫切需要更好的表达和研究整体膜蛋白的方法，但很少有系统的研究来确定导致无法恢复高水平活性蛋白的特征。这显然与膜蛋白结构的缺乏直接相关。最近对一类重要的哺乳动物膜蛋白——g蛋白偶联受体的异源表达进行了综述，列出了大肠杆菌、酵母、昆虫细胞和哺乳动物系统中的表达水平。在165例报道的异源GPCR表达研究中，只有2例在纯化尝试之前产生的蛋白水平大于1mg / 5l。为了本提案的目的，最小表达目标将是1 mg/L，其中在任何表达系统中产生100 mg或更多达到理想情况。为什么膜蛋白的过表达如此之低？十年前，表达的尝试较少，许多人相信对表达系统的实证研究将证明可以找出原因。然而，早在1995年，Grisshammer和Tate就提出，只有当我们了解膜蛋白如何折叠以及哪些细胞蛋白参与了天然环境中成功折叠时，膜蛋白过表达系统才会取得进展。这种情况在近十年来几乎没有改变。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的检查，才能改善过表达。这是这个子项目的重点。本子项目的目标有两个方面：确定膜蛋白在两种主要表达系统中的表达限制，并根据需要重新设计蛋白质和表达系统以改善表达。

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. This is clearly directly related to the paucity of membrane protein structures. A recent review of heterologous expression of an important class of mammalian membrane proteins, the G-protein coupled receptors, tabulates expression levels in E. coli, yeast, insect cell, and mammalian systems. Of 165 reported studies of heterologous GPCR expression, only 2 examples yielded protein at levels greater than 1 mg/5 L, prior to purification attempts. For the purposes of this proposal, a minimal expression goal will be 1 mg/L, where production of 100 mg or more in any expression system reaches an ideal case. Why has overexpression of membrane proteins been so low? A decade ago, expression attempts were fewer, and many believed that an empirical study of expression systems would prove to identify the cause. However, as early as 1995, Grisshammer and Tate suggested that advances in overexpression systems of membrane proteins would only result when we understood how membrane proteins fold and which cellular proteins were involved in successful folding in the native environment. This situation is virtually unchanged in almost a decade. 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 this subproject. The goals of this subproject are two-fold: 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.