PROTEIN ENGINEERED GLUCOSE SENSOR

葡萄糖传感是一项关键的医疗保健需求。 初级感测 已经用于监测血糖的部分是 葡萄糖氧化酶 不幸的是，这项技术需要一个样本 新鲜的血液被抽走。最后，传感策略可以 与闭环胰岛素输送系统连接将需要 留置传感器葡萄糖结合蛋白伴刀豆球蛋白A已经被发现， 广泛研究其在这种系统中的潜在用途，但因此 远远没有工作。 显然，需要采取不同的办法。 这里我们 建议利用葡萄糖-半乳糖 结合蛋白（GGBP）。大肠杆菌结合葡萄糖， 结合时的构象变化。 的最常见技术 将蛋白质转化为荧光生物传感器是通过随机地 用荧光染料标记。 这种随机的缺点是 技术有很多，但最重要的是，标签可能是 放置在对环境变化不敏感的地点， 我们希望检测。我们项目的新奇在于我们将- 特异性标记的GGBP，通过定点诱变， 在环境中对葡萄糖水平的最大响应位置， 蛋白质活性的最小扰动。 一种荧光供体和一种 吸收受体染料将连接到半胱氨酸突变， 蛋白质的182和26位。这些氨基酸位于 GGBP分子的相反结构域。 在没有葡萄糖的情况下，GGBP 可绕其轴自由扭转，从而允许供体和受体 染料以足够接近荧光共振能量转移 （FRET）.在葡萄糖结合后，GGBP呈现更紧凑和更刚性的结构。 结构，其中供体和受体染料面向结构的相对侧。 分子。 结果是FRET随着增加而减少。 葡萄糖浓度。 FRET的程度与 通过相位测量的供体荧光团的平均衰减时间- 调制方式 我们组建了一支精通分子生物学、发酵和 放大，光谱学和蛋白质工程来解决这个问题。 我们相信可以制造出上级的无试剂葡萄糖传感器， 可能会对实现一个 留置葡萄糖监测系统。

Glucose sensing is a critical health care need. The primary sensing moiety that has been used for monitoring blood glucose has been the enzyme glucose oxidase. Unfortunately, this technique requires a sample of fresh blood to be drawn. Ultimately, sensing strategies that can be interfaced to closed-loop insulin delivery systems will require an indwelling sensor. The glucose binding protein Concanavalin A has been extensively studied for its potential use in such a system, but has thus far failed to work. Clearly, a different approach is needed. Here we propose to take advantage of the ability of the glucose-galactose binding protein (GGBP) from E. coli to bind glucose and undergo dramatic conformational changes upon binding. The most common technique for transforming a protein into a fluorescent biosensor is by randomly labeling it with fluorescent dyes. The disadvantages of this random technique are numerous, but the most important is that the label may be placed at a site that is not sensitive to the environmental changes that we wish to detect. The novelty of our project is that we will site- specifically label GGBP, by site-directed mutagenesis at just the right positions for maximum response to glucose levels in the environment with minimum perturbation of protein activity. A fluorescent donor and an absorbing acceptor dye will be attached to cysteine mutations at positions 182 and 26 of the protein. These amino acids are located at opposite domains of the GGBP molecule. In the absence of glucose, GGBP is free to twist about its axis thereby allowing the donor and acceptor dyes to get close enough for fluorescence resonance energy transfer (FRET). Upon glucose binding, GGBP assumes a more compact and more rigid structure, with the donor and acceptor dyes facing opposite sides of the molecule. The result is a decrease in FRET with increasing concentrations of glucose. The degree of FRET is correlated to the average decay time of the donor fluorophore measured by the phase- modulation method. We have assembled a team skilled in molecular biology, fermentation and scaleup, spectroscopy and protein engineering to tackle this problem. We believe that a superior reagentless glucose sensor can be made and can have a significant impact on the ability to move towards an indwelling glucose monitoring system.