A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.
A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.
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DOI：
10.1016/j.ab.2012.01.034
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发表时间：
2012-05-01
影响因子：
2.9
通讯作者：
Wright GJ
中科院分区：
文献类型：
--
作者：
Sun Y;Gallagher-Jones M;Barker C;Wright GJ
低亲和力细胞外蛋白相互作用对于细胞识别过程至关重要，但现有的检测它们的方法在规模上受到限制，使得全基因组相互作用筛选在技术上具有挑战性。为了解决这一问题，我们在这里报告的微型化的AVEXIS（亲合力为基础的细胞外相互作用屏幕）测定通过使用蛋白质微阵列技术。为了实现这一目标，我们开发了蛋白质标签和样品制备方法，使数百个在哺乳动物细胞中表达的重组蛋白质的平行纯化。我们对一组已知的定量受体-配体对进行了基于蛋白质微阵列的检测，并表明它足够灵敏，可以检测到这类相互作用中典型的非常弱的相互作用。规模的增加使得能够针对微阵列上的固定化蛋白质的稀释系列进行相互作用筛选，使得能够观察饱和结合行为以显示相互作用特异性，并且还能够直接从初级筛选估计相互作用亲和力。这些方法上的改进现在允许在全基因组范围内筛选新的细胞外受体-配体相互作用。
Low-affinity extracellular protein interactions are critical for cellular recognition processes, but existing methods to detect them are limited in scale, making genome-wide interaction screens technically challenging. To address this, we report here the miniaturization of the AVEXIS (avidity-based extracellular interaction screen) assay by using protein microarray technology. To achieve this, we have developed protein tags and sample preparation methods that enable the parallel purification of hundreds of recombinant proteins expressed in mammalian cells. We benchmarked the protein microarray-based assay against a set of known quantified receptor–ligand pairs and show that it is sensitive enough to detect even very weak interactions that are typical of this class of interactions. The increase in scale enables interaction screening against a dilution series of immobilized proteins on the microarray enabling the observation of saturation binding behaviors to show interaction specificity and also the estimation of interaction affinities directly from the primary screen. These methodological improvements now permit screening for novel extracellular receptor–ligand interactions on a genome-wide scale.
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影响因子：
4.8
作者：
Dustin, ML;Golan, DE;van der Merwe, PA
通讯作者：
van der Merwe, PA
影响因子：
--
作者：
Letarte, Michelle;Voulgaraki, Despina;Barclay, A. Neil
通讯作者：
Barclay, A. Neil
影响因子：
7.3
作者：
Humphries JD;Byron A;Bass MD;Craig SE;Pinney JW;Knight D;Humphries MJ
通讯作者：
Humphries MJ
影响因子：
5.4
作者：
Jiang, Lei;Barclay, A. Neil
通讯作者：
Barclay, A. Neil
影响因子：
64.8
作者：
Gavin, AC;Bösche, M;Superti-Furga, G
通讯作者：
Superti-Furga, G