Development of Genetically-Encoded Glucose Sensors

描述(申请人提供)：在大多数生物体中，包括动物、植物和细菌，葡萄糖被用作能量来源，用于能量存储和合成必要的生物分子。在正常情况下和疾病状态下，血糖测量对于阐明葡萄糖在细胞和亚细胞水平上分布的基本特征至关重要。这一点特别重要，因为在糖尿病这一人类最常见的慢性病中，葡萄糖代谢的变化(在细胞水平上没有得到很好的了解)是最重要的因素。目前，还没有一种适合于测量细胞内葡萄糖的传感器，可用于临床和工业应用的传感器往往不能令人满意。这项建议提出了一种新的方法来开发葡萄糖传感器，基于使用自体荧光蛋白(在没有辅助因子的情况下会发光的蛋白质)和葡萄糖激酶(己糖激酶IV)的葡萄糖结合特性，葡萄糖激酶是胰腺的葡萄糖传感器蛋白。最有用的自体荧光蛋白来自水母Aequoria Victoria的绿色荧光蛋白和软骨瘤属珊瑚的红色荧光蛋白(红色荧光蛋白)。我们的计划是开发一种传感器，它将：1)适合于“体外”测量葡萄糖浓度；2)基因编码，用于定位特定的细胞和亚细胞隔间，以及转基因动物的特定细胞和组织。该传感器还可以被评估为糖尿病患者持续微创血糖监测系统的主干。我们计划通过将葡糖激酶融合到自体荧光蛋白上来设计葡萄糖传感器。我们将纯化在大肠杆菌中表达的传感器蛋白，分析它们的性质(荧光特性、对葡萄糖的响应、特异性)，并测试它们在测量活细胞中葡萄糖的有效性。虽然该项目的目标是获得一种用于细胞测量的传感器，以促进在正常和疾病条件下的基础研究，但新传感器可能适用于工业(例如，果汁中的葡萄糖测量)和医疗(监测糖尿病中的血糖)用途。

DESCRIPTION (provided by applicant): In most living organisms, including animals, plants and bacteria, glucose is employed as energy source, for energy storage and in the synthesis of essential biological molecules. Glucose measurements are critical to elucidate the basic characteristics of glucose distribution at the cellular and subcellular level under normal conditions and in disease states. This is particularly important because in diabetes mellitus, one of the most common chronic diseases in humans, alterations in glucose metabolism (not well understood at the cell level) are the most important factor. Currently, there is no glucose sensor suitable for measuring glucose in cells and the sensors available for clinical and industrial applications are frequently unsatisfactory. This proposal presents a novel approach to develop glucose sensors based on the use of autofluorescent proteins (proteins that fluoresce in the absence of cofactors) and the glucose binding properties of the enzyme glucokinase (hexokinase IV), the glucose sensor protein of the pancreas. The most useful autofluorescent proteins derive from the green fluorescent protein from the jellyfish Aequoria victoria and the red fluorescent protein from a coral of the Discosoma genus (red fluorescent protein). Our plan is to develop a sensor that will be: 1) Suitable for "in vitro" measurements of glucose concentration, and 2) Genetically-encoded for targeting to specific cells and subcellular compartments, as well as specific cells and tissues of transgenic animals. The sensor can also be evaluated as the backbone for a continuous minimally invasive glucose monitoring system in diabetic patients. We plan to engineer glucose sensors by fusion of glucokinase to autofluorescent proteins. We will purify the sensor proteins expressed in E. coil, analyze their properties (fluorescence properties, response to glucose, specificity) and test their usefulness for measuring glucose in living cells. Although the goal of the project is to obtain a sensor for measurements in cells, to foster basic research under normal and disease conditions, potentially the new sensors can be adapted to industrial (e.g., glucose measurements in fruit juices) and medical (monitoring glycemia in diabetes mellitus) uses.