A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells
A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells
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DOI：
10.1158/1078-0432.ccr-10-0548
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发表时间：
2010-08-01
影响因子：
11.5
通讯作者：
Ohba, Yusuke
中科院分区：
文献类型：
--
作者：
Mizutani, Tatsuaki;Kondo, Takeshi;Ohba, Yusuke
目的：为了开发一种新的诊断方法来评估慢性髓性白血病（CML）患者的药物疗效，我们制造了一种生物传感器，可以利用荧光共振能量转移（FRET）原理评估活细胞中BCR-ABL激酶的活性。实验设计：为了开发基于FRET的生物传感器，我们使用了BCR-ABL最具特征的底物CrkL，并设计了一种蛋白质，将CrkL夹在YFP变体Venus和增强的青色荧光蛋白之间，使CrkL在SH2结构域与磷酸化酪氨酸（Y207）的分子内结合提高了FRET效率。在与Western blotting和流式细胞术等现有方法进行比较后，对该生物传感器的性能进行了评估，并在CML患者细胞中检测了BCR-ABL活性及其对药物的反应。结果：优化后获得的生物传感器在测量BCR-ABL活性和伊马替尼抑制BCR-ABL活性方面具有比现有技术更高的灵敏度。由于其高灵敏度，这种生物传感器可以准确地测量相对较小细胞数量的BCR-ABL活性，并且还可以检测
Purpose: To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (CML) patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET).Experimental Design: To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with established methods including Western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells.Results: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect