Activation of circularly permutated β-lactamase tethered to antibody domains by specific small molecules.
Activation of circularly permutated β-lactamase tethered to antibody domains by specific small molecules.
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DOI：
10.1021/bc1004125
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发表时间：
2011-03
影响因子：
4.7
通讯作者：
M. Kojima;Hiroto Iwai;Jinhua Dong;Shean-Lee Lim;S. Ito;K. Okumura;M. Ihara;H. Ueda
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文献类型：
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作者：
M. Kojima;Hiroto Iwai;Jinhua Dong;Shean-Lee Lim;S. Ito;K. Okumura;M. Ihara;H. Ueda
酶的活性受其底物或效应物的调节通常称为变构。然而，它被认为很难改变其效应特异性，尽管它作为一个敏感的分子传感器的潜在效用。为此，我们制备了由抗体可变区Fv和环状排列的TEM-1 β-内酰胺酶cpBLA组成的融合蛋白。构建了两种抗原特异性不同的表达载体，将cpBLA插入到单链抗体的连接区，分别与骨相关疾病标志物骨钙素（BGP）C-端肽和类烟碱杀虫剂吡虫啉（ICP）特异性结合。在活性位点附近具有新末端的cpBLA在与其同源抗原结合时被激活，这是由于结合抗原稳定了拴系的Fv。结果，两种Fv-cpBLA都显示出特异性抗原结合以及抗原诱导的催化活性增强。此外，E.在含有氨苄青霉素的培养基中，表达抗ICP的Fv-cpBLA的大肠杆菌细胞显示ICP浓度依赖性生长。该系统还用于选择赋予更快生长的Fv-cpBLA接头突变体。这将是第一个基于抗体的小分子指示酶。
Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 β-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.