title: Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection.
abstract: 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).
authors.affiliation: IRTA, Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Garibo
authors.forename: Eric
authors.initials: E
authors.lastname: Devic
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Marty
authors.forename: Jorge
authors.initials: J
authors.lastname: Diogène
authors.forename: Idoia
authors.initials: I
authors.lastname: Unzueta
authors.forename: María
authors.initials: M
authors.lastname: Blázquez
authors.forename: Mònica
authors.initials: M
authors.lastname: Campàs
mesh_terms: D000818:Animals; D019149:Bioreactors; D002846:Chromatography, Affinity; D003124:Colorimetry / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D059346:Magnets; D008387:Marine Toxins / Q000032:analysis* / Q000378:metabolism; D019319:Okadaic Acid / Q000032:analysis* / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012758:Shellfish
chemical_list: D004800:Enzymes, Immobilized; D008387:Marine Toxins; D011993:Recombinant Fusion Proteins; D019319:Okadaic Acid; D006639:Histidine; D054648:Protein Phosphatase 2