Development of a GABA Enzyme for Biosensor and Point-of-Care Applications

 描述（由申请人提供）：作为大脑中最重要的抑制性神经递质，对γ-氨基丁酸（GABA）释放的影响的详细理解仍然是难以捉摸的。GABA浓度的测量是一个困难的过程。微透析是自由移动动物脑中GABA采样的当前标准，但存在时间分辨率低和需要劳动密集型分析方法的问题。相比之下，GABA的直接感测，包括生物传感器的方式，提供秒的时间分辨率，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感兴趣的分析物。将GABA酶促转化为可转导信号的最新技术是三种单独的酶或抗体包埋在纳米颗粒内的连续活性。对于CNS和全身性GABA传感应用，需要单个GABA氧化酶。目前还没有这样的GABA氧化酶。为了开发这种酶，品尼高将与堪萨斯大学的两名顶尖科学家组成的跨学科小组合作。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。该团队已经克隆、表达纯化并表征了具有一定GABA活性的氧化酶（wt-pUUB-Ox）。在第一阶段，我们将使用这种氧化酶作为起始支架来进化出真正的GABA氧化酶。在第1阶段结束时，我们将获得一种氧化酶，其GABA活性相对于wt-pUUB-Ox提高了10倍至50倍，并且第II阶段的氧化酶活性和稳定性的明确途径适用于生理学相关GABA浓度的特定测量。这种进化的GABA氧化酶将在单一反应步骤中氧化GABA以产生过氧化氢作为副产物。GABA氧化酶可以作为新的监测模式的基础，否则是不可能的。到第二阶段结束时，将有两种市售产品可供使用。首先，GABA生物传感器用于实时测量临床前模型大脑中GABA的生理相关水平，其次，GABA氧化酶用于各种诊断和即时护理设备。

 DESCRIPTION (provided by applicant): 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, provides 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 three separate enzymes or antibodies entrapped within nanoparticles. For CNS and systemic GABA sensing applications, a single GABA oxidase enzyme is necessary. No such oxidase enzyme for GABA is currently available. To develop this enzyme, 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. This team has already cloned, expressed purified and characterized an oxidase enzyme (wt-pUUB-Ox) with some GABA activity. During Phase I, we will use this oxidase enzyme as a starting scaffold to evolve a true GABA oxidase enzyme. At the end of Phase 1, we will have an oxidase enzyme with a 10x - 50x improvement in GABA activity relative to wt-pUUB-Ox, and a clear path for Phase II to oxidase activity and stability suitable for the specific measurement of physiologically relevant GABA concentrations. This evolved GABA oxidase enzyme will, in a single reaction step, oxidize GABA to produce hydrogen peroxide as a byproduct. The GABA oxidase enzyme can be used as the basis for new monitoring paradigms that would otherwise be impossible. 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 models and second, a GABA oxidase enzyme for use in a variety of diagnostic and point-of-care devices.