Development of a GABA Enzyme for Biosensor Applications

开发用于生物传感器应用的 GABA 酶

• 批准号: 9464830
• 负责人: DAVID A JOHNSON
• 金额: $ 56.93万
• 依托单位: PINNACLE TECHNOLOGY, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9464830
• 关键词: Active Sites; Address; Affinity; Alzheimer' s Disease; Americas; Amino Acids; Animal Model; Animals; Antibodies; Anxiety; Biosensor; Brain; Cells; Collection; Complex; Core Facility; Crystallization; DNA; DNA-Directed RNA Polymerase; Development; Devices; Diagnostic; Directed Molecular Evolution; Disease; Disease model; Electrodes; Electronics; Elements; Ensure; Environmental Risk Factor; Enzymes; Exhibits; Genes; Growth; Hospitals; Huntington Disease; Immobilized Enzymes; Individual; Kansas; Life; Malignant Neoplasms; Measurable; Measurement; Methods; Microdialysis; Modality; Modeling; Modification; Monitor; Mus; Mutation; Neuraxis; Neurotransmitters; Nitrogen; Oxidases; Parkinson Disease; Phase; Physiological; Play; Process; Production; Productivity; Progress Reports; Property; Protein Engineering; Proteins; Quality of life; Rattus; Reaction Time; Research; Resolution; Role; Sales; Sampling; Scientist; Seizures; Signal Transduction; Source; Structure; Substrate Specificity; System; Technology; Temperature; Time; Universities; Visiting Nurse; Work; ascorbate; brain circuitry; design; economic cost; enzyme immobilization; enzyme structure; experience; gamma-Aminobutyric Acid; improved; improved functioning; in vivo; interest; meetings; monitoring device; mutant; nanoparticle; novel; nursing home length of stay; point of care; pre-clinical; professor; protein folding; temporal measurement; therapy development; tool

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.

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）释放的影响仍然难以捉摸。GABA的测量 集中是一个艰难的过程。微透析是GABA采样的当前标准， 自由移动的动物的大脑，但患有低时间分辨率和需要劳动力 深入分析方法。相比之下，GABA的直接感知，通过包括 生物传感器，将提供每秒的时间分辨率，而不需要额外的后， 分析.然而，生物传感器和其他监测设备需要酶来处理分析物 感兴趣将GABA酶促转化为可转导信号的最新技术是 纳米颗粒内包埋的多种酶或抗体的连续活性。脑与 系统性GABA传感应用中，一个单一的GABA氧化酶是必要的。无此类氧化酶 GABA的酶是目前可用的。 该提议通过设计单一GABA特异性氧化酶来解决该问题， 该生物识别元件适用于制作GABA生物传感器。在第一阶段，我们 鉴定，分离，纯化，克隆，结晶和模拟的活性位点的结构， 甲基氨基丁酸氧化酶（MGOX）。我们建立了一个定向进化系统（KJ109）， 只有当GABA作为氮源时才能生长，我们证明了初步的 MGOX酶可用于生物传感器以检测GABA。本项目第二阶段有两个 主要解决的问题是：（1）对MGOX进行修饰，提高其在生理pH下的kcat （~pH 7.4）;和（2）修饰MGOX以增加检测生理相关GABA的灵敏度 浓度的Pinnacle将与由两位顶尖科学家组成的跨学科小组合作 堪萨斯大学。Mark Richter教授是蛋白质工程和蛋白质折叠方面的专家， Philip Gao博士是蛋白质生产核心设施的主任。到第二阶段结束时， 将可获得市售产品。首先，用于实时测量的GABA生物传感器 临床前动物模型大脑中GABA的生理相关水平，第二， GABA氧化酶，用于各种诊断和即时护理器械。