Engineered alkaline phosphatases as biosensors

作为生物传感器的工程碱性磷酸酶

• 批准号: 6598461
• 负责人: ICHIRO MATSUMURA
• 金额: $ 26.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6598461
• 关键词: Bacillus anthracis; Escherichia coli; Yersinia pestis; alkaline phosphatase; antibody; aspartic endopeptidases; bioengineering /biomedical engineering; biohazard detection; biosensor device; biotechnology; bioterrorism /chemical warfare; enzyme activity; hemagglutinin; metalloendopeptidases; peptide library; protein engineering; technology /technique development

DESCRIPTION (provided by applicant): Bacillus anthracis spores are currently detected by established but slow microbiological test procedures. The development of faster, cheaper and higher-throughput detection methods would enable more effective responses to bio-terrorism and natural infectious disease outbreaks. Our goal is to engineer a reporter enzyme so that is inactive until it encounters a pathogen marker. Our design strategy is to imitate the twostep natural evolution of "intrasteric" regulation. We have already generated a variant of the Escherichia coil beta-galactosidase (BGAL) that is specifically activated 5.7-fold when co-expressed with the human immuno-deficiency (HIV) protease. We believe that the E. coil alkaline phosphatase (AP) has even greater potential as a biosensor, and propose studies with the following specific aims: 1. to isolate effector-dependent AP variants with greater response to the HIV protease (>570% activation) and more robust enzyme activities. 2. to "re-program" the best biosensor so that its activity becomes dependent upon the B. anthracis Lethal Factor (LF), the anti-influenza hemagglutinin (HA) antibody, or the anti-Yersinia pestis F1 antibody. 3. to array biosensors that respond to different effectors upon a chip for the rapid detection of pathogen markers. The biosensors generated in this study will streamline disease diagnosis by supplanting time-consuming and expensive immunoassays. These experiments will test the feasibility of our evolutionary hypothesis and demonstrate the utility of novel protein engineering techniques.

描述(申请人提供)：炭疽芽胞目前是通过已建立但缓慢的微生物测试程序检测出来的。开发更快、更便宜和更高通量的检测方法将使人们能够更有效地应对生物恐怖主义和自然传染病的爆发。我们的目标是设计一种报告酶，使其在遇到病原体标记之前处于不活跃状态。我们的设计策略是模仿“空间内”监管的两步自然演变。我们已经产生了一种大肠杆菌线圈β-半乳糖苷酶(BGAL)的变体，当与人类免疫缺陷(HIV)酶共表达时，该变体被特异性激活5.7倍。我们认为大肠杆菌碱性磷酸酶(AP)作为生物传感器具有更大的潜力，并提出了以下具体目标的研究：1.分离对HIV蛋白酶有更强反应(&gt；570%激活)和更强酶活性的效应依赖型AP变体。2.对最好的生物传感器重新编程，使其活性依赖于炭疽杆菌致死因子(LF)、抗流感血凝素(HA)抗体或抗鼠疫F1抗体。3.在芯片上排列响应不同效应器的生物传感器，以快速检测病原体标志物。这项研究中产生的生物传感器将通过取代耗时和昂贵的免疫分析来简化疾病诊断。这些实验将检验我们进化假说的可行性，并展示新的蛋白质工程技术的实用性。