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Glucose Sensing Based on an ABC Transporter System

基于 ABC 转运系统的葡萄糖传感

基本信息

批准号：
6825700
负责人：
LEAH TOLOSA
金额：
$ 14.12万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE COUNTY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-15 至 2006-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6825700
关键词：
Glucose Sensing Based ABC Transporter

项目摘要

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================

超出提供的空间。糖尿病是一种慢性疾病，随着美国人口老龄化，它正接近流行病的比例。目前还没有确定的治愈方法，但患者可以通过健康的生活方式、新的更有效的药物和持续的血糖测量来过上接近正常的生活。然而，现有的血糖监测系统不方便，价格昂贵，而且一天只能监测几次。最近FDA批准的连续血糖监测仪的使用有限，并且由于葡萄糖氧化酶的降解而产生漂移，葡萄糖氧化酶是传感机制的核心。包括我们自己在内的几个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品，并取得了一定程度的成功。这项提议寻求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，因为它们发生在自然界中。这种方法将利用大肠杆菌的葡萄糖渗透酶系统，即由周质葡萄糖结合蛋白(GBP或MglB)组成的ABC转运体，以及两个拷贝的膜跨越蛋白(Mglc)和ATPase(MglA)。具体地说，我们将：1.利用重组技术在大肠杆菌中高效表达和分离这些蛋白。2.用适当的FRET供体/受体对标记GBP和MGLC。3.将mglc和mglA重组到聚合物脂质体或膜中。4.通过测量MGIB/mglc/mglA(微摩尔葡萄糖敏感度)和mglc/mglA(毫摩尔葡萄糖敏感度)的FRET来评价血糖反应。5.通过设计缺乏ATPase活性但对葡萄糖结合有反应的MglA突变体，设计了一种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，用于活体监测。仪器、化学和固定化技术已经到位。所需要的只是一种专门针对葡萄糖的信号转换器，具有正确的灵敏度，并且长期可靠。这项提案寻求为基于ABC转运体系统的新型生物传感器的开发提供资金，这可能为更广泛的生物医学重要分析物铺平道路。表演网站========================================Section End===========================================