Engineering a regulatable enzyme for homogeneous immunoassays
Engineering a regulatable enzyme for homogeneous immunoassays
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DOI：
10.1038/5243
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发表时间：
1999-01-01
影响因子：
46.9
通讯作者：
Fastrez, J
中科院分区：
文献类型：
--
作者：
Legendre, D;Soumillion, P;Fastrez, J
我们已经设计了噬菌体展示TEM-1 β-内酰胺酶，以产生可用于均相免疫测定的酶，因为它们的活性可以通过与针对不相关蛋白质产生的单克隆抗体（Mab）结合来调节。将随机肽文库遗传插入三个环中以产生具有Mab结合位点的杂合酶。选择插入点以足够接近活性位点，使得复合物形成可影响活性。由β-内酰胺酶活性提供的抗生素抗性用于选择编码活性酶的克隆。在针对前列腺特异性抗原（PSA）的固定化单克隆抗体上或在链霉亲和素上的活性文库的生物淘选产生了具有这些蛋白质的结合位点的酶。它们的活性可以通过单克隆抗体或链霉亲和素结合来调节。络合物的离解常数在10（-9）~ 10（-6）M范围内。在竞争性测定中，可以在10（-9）M的最低浓度下检测到PSA。单克隆抗体识别模拟表位，因为在调节克隆中的插入片段和PSA序列片段之间没有发现序列相似性。该方法可以被开发以产生用于检测溶液中的分析物的信号分子，而无需鉴定表位。
We have engineered the phage displayed TEM-1 beta-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein. Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs. Insertion points were chosen to be close enough to the active site that complex formation could affect the activity. The antibiotic resistance provided by the beta-lactamase activity was used to select the clones encoding active enzymes. Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins. Their activity could be regulated by Mab or streptavidin binding. The dissociation constants of the complexes are in the 10(-9) to 10(-6) M range. In a competitive assay, PSA could be detected at a minimal concentration of 10(-9) M. The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence. The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.