Engineered alkaline phosphatases as biosensors

作为生物传感器的工程碱性磷酸酶

基本信息

批准号：
6721456
负责人：
ICHIRO MATSUMURA
金额：
$ 26.6万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-01 至 2006-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6721456
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

项目摘要

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（由申请人提供）：目前通过已建立的但缓慢的微生物测试程序来检测炭疽芽孢杆菌孢子。开发更快、更便宜和更高通量的检测方法将使我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非活性状态。我们的设计策略是模仿“星际”调节的两步自然演化。我们已经生成了大肠杆菌 β-半乳糖苷酶 (BGAL) 的变体，当与人类免疫缺陷 (HIV) 蛋白酶共表达时，该变体被特异性激活 5.7 倍。我们相信大肠杆菌碱性磷酸酶 (AP) 作为生物传感器具有更大的潜力，并提出了具有以下具体目标的研究： 1. 分离对 HIV 蛋白酶具有更大反应的效应依赖性 AP 变体（>570% 激活））和更强大的酶活性。 2.“重新编程”最好的生物传感器，使其活性依赖于炭疽芽孢杆菌致死因子（LF）、抗流感血凝素（HA）抗体或抗鼠疫耶尔森氏菌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记。这项研究中产生的生物传感器将通过取代耗时且昂贵的免疫测定来简化疾病诊断。这些实验将测试我们进化假设的可行性，并证明新型蛋白质工程技术的实用性。