Expression and Purification of PI3 Kinase α and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay
Expression and Purification of PI3 Kinase α and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay
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DOI：
10.1177/1087057108326079
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发表时间：
2008-12-01
影响因子：
--
通讯作者：
Issinger, Olaf-Georg
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文献类型：
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作者：
Boldyreff, Brigitte;Rasmussen, Tine L.;Issinger, Olaf-Georg
磷酸肌苷-3激酶是药物开发的重要靶点，因为PI3激酶信号通路中的许多蛋白质在人类癌症中发生突变、过度激活或过度表达。在Sf9昆虫细胞中，作者共表达了人类Ia类PI3激酶p110 α催化结构域与n端His-tag和p85 α调节结构域。通过镍亲和层析纯化了p110a和p85a配合物。作者建立了三磷酸腺苷（ATP）消耗测定法来测定p110 α /p85 α的活性。通过测试不同的脂质作为底物，以及不同的激酶和脂质浓度，对该方法进行了优化。此外，他们分析了p110 α /p85 α的自磷酸化，并确定了wortmannin（一种已知的PI3激酶抑制剂）的IC50。测定wortmannin的IC50为7 nM。在选择的底物中，在50 μ m的浓度下，磷脂酰肌醇-4,5-二磷酸被证明是最好的底物。p110 α / p85a在p85 α亚基上发生了最显著的自磷酸化。然而，在存在脂质底物的情况下，自磷酸化可以忽略不计。同时，使用alphasgreen技术的第二种检测格式被优化以测量PI3激酶活性。所使用的两种分析格式都应适用于PI3激酶抑制剂的高通量筛选。（Journal of biomolmolecular Screening 2008: 1035-1040）
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 alpha catalytic domain with an N-terminal His-tag and the p85 alpha regulatory domain in Sf9 insect cells. The complex consisting of p110a and p85a was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110 alpha/p85 alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110 alpha/p85 alpha 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 mu M. p110 alpha/ p85a underwent autophosphorylation most prominently at the p85 alpha 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)