Glucose Sensing Based on an ABC Transporter System

基于 ABC 转运系统的葡萄糖传感

• 批准号: 6561157
• 负责人: LEAH TOLOSA
• 金额: $ 11.02万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2005-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561157
• 关键词: Escherichia coli; binding proteins; biosensor device; biotechnology; blood glucose; diabetes mellitus; fluorescence resonance energy transfer; liposomes; membrane reconstitution /synthesis; membrane transport proteins; molecular cloning; recombinant proteins

DESCRIPTION (provided by applicant): Diabetes is a chronic disease that is approaching epidemic proportions with the ageing of the US population. At present there is no established cure, but patients can lead near-normal lives with a healthy lifestyle, new and more effective drugs and constant blood glucose measurements. However, available glucose monitoring systems are inconvenient, costly and can be done only a few times during the day. Recent continuous glucose monitors approved by the FDA have limited use and suffer from drift due to the degradation of the glucose oxidase that is the heart of the sensing mechanism. Several groups, including our own, have investigated other glucose binding proteins as alternative to glucose oxidase with some degree of success. This proposal seeks to go beyond single proteins as glucose sensors, rather we will look at glucose-induced protein-protein interactions as they occur in nature. This approach will utilize the glucose permease system of E. coli, an ABC transporter comprised of the periplasmic glucose binding protein (GBP or MglB), and two copies each of a membrane spanning protein (MglC) and an ATPase (MglA). Specifically, we will: 1. Overexpress and isolate these proteins in E. coli by recombinant techniques. 2. Label GBP and MglC with appropriate FRET donor/acceptor pairs. 3. Reconstitute MglC and MglA in polymeric liposomes or membranes. 4. Evaluate glucose response by measuring FRET in MgIB/MglC/MglA (micromolar glucose sensitivity) and MglC/MglA (millimolar glucose sensitivity). 5. Devise a reagentless glucose sensor by designing MglA mutants that lack ATPase activity but respond to glucose binding. The long-term goal is to develop a low-cost optical glucose sensor for in vivo monitoring. Instrumentation, chemical and immobilization technologies are already in place. All that is needed is a signal transducer that is specific for glucose, has the right sensitivity and is reliable for long periods. This proposal seeks funding for the development of a new type of biosensor based on the ABC transporter system that may pave the way for a broader range of biomedically important analytes.

描述（由申请人提供）： 糖尿病是一种慢性疾病，随着美国人口的老龄化，它正接近流行病的程度。目前还没有确定的治疗方法，但患者可以通过健康的生活方式，新的和更有效的药物和恒定的血糖测量过上接近正常的生活。然而，可用的葡萄糖监测系统是不方便的、昂贵的，并且在一天中只能进行几次。最近由FDA批准的连续葡萄糖监测器具有有限的使用，并且由于作为感测机制的核心的葡萄糖氧化酶的降解而遭受漂移。包括我们自己在内的几个研究小组已经研究了其他葡萄糖结合蛋白作为葡萄糖氧化酶的替代物，并取得了一定程度的成功。该提案旨在超越单一蛋白质作为葡萄糖传感器，而是我们将研究葡萄糖诱导的蛋白质-蛋白质相互作用，因为它们在自然界中发生。该方法将利用E.大肠杆菌中，ABC转运蛋白包括周质葡萄糖结合蛋白（GBP或MglB），以及各两个拷贝的跨膜蛋白（MglC）和ATP酶（MglA）。具体来说，我们将：1。在E.大肠杆菌中进行重组。2.用适当的FRET供体/受体对标记GBP和MglC。3.在聚合物脂质体或膜中重构MglC和MglA。4.通过测量MgIB/MglC/MglA（微摩尔葡萄糖敏感性）和MglC/MglA（毫摩尔葡萄糖敏感性）中的FRET来评价葡萄糖响应。5.通过设计缺乏ATP酶活性但响应葡萄糖结合的MglA突变体来设计无试剂葡萄糖传感器。 长期目标是开发一种低成本的光学葡萄糖传感器用于体内监测。仪器、化学和固定技术已经到位。所需要的只是一个专门针对葡萄糖、具有正确灵敏度且长期可靠的信号传感器。该提案旨在为开发基于ABC转运系统的新型生物传感器提供资金，该系统可能为更广泛的生物医学重要分析物铺平道路。