Characterization and activity determination of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae.
Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 μg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 μmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at -20 °C.
Departamento de Fisiología, Facultad Veterinaria, Lugo, Spain. ja.rubiolo@usc.es
J A
JA
Rubiolo
H
H
López-Alonso
A
A
Alfonso
F V
FV
Vega
M R
MR
Vieytes
L M
LM
Botana
D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007814:Larva / Q000235:genetics; D009036:Moths / Q000235:genetics*; D054648:Protein Phosphatase 2 / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D021122:Protein Subunits; D054648:Protein Phosphatase 2