Engineering regulable Escherichia coli β-galactosidases as biosensors for anti-HIV antibody detection in human sera
Engineering regulable Escherichia coli β-galactosidases as biosensors for anti-HIV antibody detection in human sera
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DOI：
10.1074/jbc.m104704200
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发表时间：
2001-10-26
影响因子：
4.8
通讯作者：
Villaverde, A
中科院分区：
文献类型：
--
作者：
Ferrer-Miralles, N;Feliu, JX;Villaverde, A
工程化的肽展示酶的活性通过与特异性抗肽抗体结合来调节。定量抗体检测系统的这一新概念允许建立用于快速诊断传染病的检测试剂盒。为了建立一种快速、均质的检测人类免疫缺陷病毒（HIV）感染的方法，我们探索了大肠杆菌β-半乳糖苷酶的两个受体位点，以适应HIV抗原肽。来自gp 41包膜糖蛋白的两个重叠表位（即P1和P2），包含在不同大小的肽中，被插入酶活性位点附近，以产生一组杂合的、具有酶活性的β-半乳糖苷酶。用两个受体位点测试产生了对单克隆抗体结合具有不同响应性的可调节酶。这些生物传感器对HIV感染者的免疫血清也很敏感。建模数据提供了深入了解的活性位点附近的结构修饰诱导肽插入，强烈影响工程蛋白的反应性，通过不同的参数，其催化性能。
The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta -galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta -galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.