Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection.
Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection.
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DOI：
10.1016/j.jbiotec.2011.11.020
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发表时间：
2012
影响因子：
4.1
通讯作者：
D. Garibo;E. Devic;J. Marty;J. Diogène;I. Unzueta;M. Blázquez;M. Campàs
中科院分区：
文献类型：
--
作者：
D. Garibo;E. Devic;J. Marty;J. Diogène;I. Unzueta;M. Blázquez;M. Campàs
本文介绍了通过基因工程获得的蛋白磷酸酶2A(PP2A)催化亚基的功能特性及其与磁性颗粒(MPS)的金属配位化学偶联，为随后建立腹泻性亲脂性海洋毒素的检测方法奠定了基础。游离酶比色法可以确定最好的酶活性稳定剂，即甘油含量为10%。该重组酶对冈田酸(LOD=2.3μg/L)和甲藻毒素-1(DTX-1)(LOD=15.2μg/L)同样敏感，且具有较高的操作稳定性，因此可以用较低的酶量进行蛋白磷酸酶抑制实验。一旦与MPS结合，PP2A催化亚基仍保持其酶活性，并可被OA抑制(LOD=30.1MPS g/L)。
This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).