DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

大肠杆菌和酵母中 GPCR 表达的决定因素

• 批准号: 7381189
• 负责人: ANNE SKAJA ROBINSON
• 金额: $ 27.27万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381189
• 关键词: DETERMINANTS; GPCR; EXPRESSION; COLI; YEAST

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.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。这显然与膜蛋白结构的缺乏有直接关系。最近综述了一类重要的哺乳动物膜蛋白--G蛋白偶联受体在大肠杆菌、酵母、昆虫细胞和哺乳动物系统中的异源表达水平。在已报道的关于异源gpcr表达的165项研究中，只有2例在纯化尝试之前，L的蛋白质含量高于1 mg/5。对于本提案的目的，最低表达目标将是1毫克/L，在任何表达系统中100毫克或更多的产量都达到理想情况。为什么膜蛋白的过度表达如此之低？十年前，表达的尝试较少，许多人认为，对表达系统的实证研究会证明这是原因。然而，早在1995年，Grisshammer和Tate就提出，只有当我们了解了膜蛋白是如何折叠的，以及哪些细胞蛋白参与了天然环境中成功的折叠时，膜蛋白过度表达系统的研究才会取得进展。这种情况在近十年来几乎没有变化。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。这是本子项目的重点。这个子项目的目标有两个：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。