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Expression and Purification of PI3 Kinase and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

KinaseDetect ApS, Odense, Denmark

Tine L. Rasmussen

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

Hans H. Jensen

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

Alexandre Cloutier

PerkinElmer BioSignal, Inc., Montreal, Canada

Lucille Beaudet

PerkinElmer BioSignal, Inc., Montreal, Canada

Philippe Roby

PerkinElmer BioSignal, Inc., Montreal, Canada

Olaf-Georg Issinger

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, ogi{at}bmb.sdu.dk

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110 catalytic domain with an N-terminal His-tag and the p85 regulatory domain in Sf9 insect cells. The complex consisting of p110 and p85 was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110/p85. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110/p85 and determined the IC₅₀ for wortmannin, a known PI3 kinase inhibitor. The IC₅₀ for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 µM. p110/p85 underwent autophosphorylation most prominently at the p85 subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. (Journal of Biomolecular Screening 2008:1035-1040)

Key Words: phosphoinositide 3-kinase • PI3K • PI3K • ATP depletion assay • AlphaScreen

This version was published on December 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 10, 1035-1040 (2008)
DOI: 10.1177/1087057108326079


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