Development of Genetically-Encoded Glucose Sensors

描述（由申请人提供）：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，用于能量储存和合成必需的生物分子。葡萄糖测量对于阐明正常情况下和疾病状态下细胞和亚细胞水平上葡萄糖分布的基本特征至关重要。这一点尤其重要，因为糖尿病是人类最常见的慢性疾病之一，葡萄糖代谢的改变（在细胞水平上还没有得到很好的理解）是最重要的因素。目前，还没有适合测量细胞内葡萄糖的葡萄糖传感器，用于临床和工业应用的传感器往往不令人满意。本提案提出了一种基于使用自身荧光蛋白（在没有辅助因子的情况下荧光的蛋白质）和葡萄糖激酶酶（己糖激酶IV）的葡萄糖结合特性（胰腺的葡萄糖传感器蛋白）来开发葡萄糖传感器的新方法。最有用的自体荧光蛋白来自维多利亚水母的绿色荧光蛋白和盘藻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)适用于“体外”测量葡萄糖浓度，2)针对特定细胞和亚细胞区室，以及转基因动物的特定细胞和组织进行遗传编码。该传感器还可作为糖尿病患者连续微创血糖监测系统的骨干。我们计划通过将葡萄糖激酶与自身荧光蛋白融合来设计葡萄糖传感器。我们将纯化E. coil中表达的传感器蛋白，分析其特性（荧光特性、对葡萄糖的响应、特异性），并测试其在活细胞中测量葡萄糖的有效性。虽然该项目的目标是获得一种用于细胞测量的传感器，以促进正常和疾病条件下的基础研究，但这种新型传感器可能适用于工业（例如，果汁中的葡萄糖测量）和医疗（监测糖尿病患者的血糖）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.