期刊
JOURNAL OF BIOTECHNOLOGY
卷 157, 期 1, 页码 89-95出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2011.11.020
关键词
Genetically engineered protein phosphatase 2A (PP2A); Protein phosphatase inhibition assay (PPIA); Magnetic particles (MPs); Ni-His affinity; Marine toxin okadaic acid (OA); Dinophysistoxin-1 (DTX-1)
资金
- INKOA Sistemas
- Universite de Perpignan-Via Domitia through the BIOKA EUROTRANSBIO
- INTERREG SUDOE IVB
- FEDER [SOE1/P1/E129 ALARMTOX, RTA2008-00084-00-00]
- INIA
- MICINN
- European Social Fund
- ALARMTOX
- Comissionat per a Universitats i Recerca of the Departament d'Innovacio, Universitats i Empresa of the Generalitat de Catalunya
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 = 2.3 mu g/L) and dinophysistoxin-1 (DTX-1) (LOD = 15.2 mu g/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 = 30.1 mu g/L). (C) 2011 Elsevier B.V. All rights reserved.
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