Development of a GABA Enzyme for Biosensor Applications

摘要 作为大脑中最重要的抑制性神经递质，详细了解 γ-氨基丁酸(GABA)释放的意义仍然难以捉摸。氨基丁酸的测定 浓缩是一个艰难的过程。微透析法是目前用于人体内GABA采样的标准 自由活动动物的大脑，但存在时间分辨率低和需要劳动的问题 密集的分析方法。相比之下，对GABA的直接感知，通过包括 生物传感器将提供一秒一秒的时间分辨率，而不需要额外的后处理 分析。然而，生物传感器和其他监测设备需要一种酶来处理分析物 感兴趣的人。将GABA酶转化为可转导信号的最先进技术是 包埋在纳米颗粒中的多种酶或抗体的顺序活性。对于大脑和 在系统的GABA传感应用中，单一的GABA氧化酶是必需的。没有这样的酶 针对GABA的酶目前已上市。 这一建议通过设计单一的GABA特定的氧化酶来解决这个问题，作为 适用于制造GABA生物传感器的生物识别元件。在第一阶段，我们 鉴定、分离、纯化、克隆、结晶和模拟ɣ-N-的活性中心结构 甲氨基丁酸氧化酶(MGOX)。我们建立了一个有向进化系统(KJ109)，其中有 只有当GABA作为氮源时才会生长，我们证明了初步的 MGOX酶可以用在生物传感器上检测GABA。对于这个项目的第二阶段，有两个 需要解决的主要问题：(1)对MGOX进行改性以提高其在生理pH下的kcat (~pH 7.4)；和(2)修饰MGOX以提高检测生理相关GABA的灵敏度 浓度。顶峰公司将与一个由两名顶尖科学家组成的跨学科小组合作 堪萨斯大学。马克·里希特教授是蛋白质工程和蛋白质折叠方面的专家， 菲利普·高博士是蛋白质生产核心设施的主任。到第二阶段结束时，两个 商业上可以买到的产品将会出现。首先，一种用于实时测量的GABA生物传感器 临床前动物模型中大脑中与生理相关的GABA水平；第二， 用于各种诊断和护理设备的GABA氧化酶。

Abstract As the most important inhibitory neurotransmitter in the brain, a detailed understanding of the implications of gamma-aminobutyric acid (GABA) release remains elusive. The measurement of GABA concentrations is a difficult process. Microdialysis is the current standard for GABA sampling in the brains of freely moving animals, but suffers from low temporal resolution and the need for labor intensive analysis methods. By contrast, the direct sensing of GABA, by modalities including biosensors, would provide second-by-second temporal resolution, without the need for additional post- analysis. However, biosensors and other monitoring devices, require an enzyme to process the analyte of interest. The state-of-the-art for the enzymatic conversion of GABA into a transducible signal is the sequential activity of multiple enzymes or antibodies entrapped within nanoparticles. For brain and systemic GABA sensing applications, a single GABA oxidase enzyme is necessary. No such oxidase enzyme for GABA is currently available. This proposal addresses this problem by designing a single GABA-specific oxidase enzyme for use as the biorecognition element that is suitable for fabrication of a GABA biosensor. During Phase I we identified, isolated, purified, cloned, crystallized and modeled the active-site structure of ɣ-N- Methylaminobutyrate oxidase (MGOX). We set up a directed evolution system (KJ109) in which there will only be growth if GABA is available as a nitrogen source, and we demonstrated that the preliminary MGOX enzyme can be used on a biosensor to detect GABA. For Phase II of this project, there are two major problems that must be solved: (1) Modification of MGOX to increase its kcat at physiological pH (~pH 7.4); and (2) Modification of MGOX to increase sensitivity to detect physiologically relevant GABA concentrations. Pinnacle will team with an interdisciplinary group of two leading scientists at the University of Kansas. Professor Mark Richter, is an expert in protein engineering and protein folding, and Dr. Philip Gao, is the Director of the Protein Production Core Facility. By the end of Phase II, two commercially available products will be available. First, a GABA biosensor for real-time measurement of physiologically relevant levels of GABA in the brain for preclinical animal models and second, a GABA oxidase enzyme for use in a variety of diagnostic and point-of-care devices.