Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments.
Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments.
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DOI：
10.1186/1472-6750-13-31
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发表时间：
2013-03-28
影响因子：
3.5
通讯作者：
Ueda H
中科院分区：
文献类型：
--
作者：
Ohmuro-Matsuyama Y;Chung CI;Ueda H
据预测，人类相互作用组包含150，000至300，000种蛋白质-蛋白质相互作用（PPI）。蛋白质片段互补分析（PCA）是检测PPI最广泛使用的方法之一，以及Förster共振能量转移（FRET）。迄今为止，基于萤火虫荧光素酶（Fluc）的PCA由于其高灵敏度、高信号背景（S/B）比和可逆响应而在体内、培养细胞和无细胞裂解物中成功应用。在这里，我们表明该测定也适用于纯化的蛋白质，具有出乎意料的快速动力学。制备了与雷帕霉素依赖性相互作用蛋白对融合的Split Fluc片段，并在E. coli系统，纯化至均一。当蛋白质用于PCA检测雷帕霉素依赖性PPI时，它们能够快速检测（~1 s）具有高S/B比的PPI。当Fn 7 -8结构域（长度为7 nm）被插入分裂Fluc和FKBP 12之间作为刚性接头时，其显示消除基于GFP突变体的FRET，它仍然显示出一些响应，表明相互作用配偶体的大小限制较少。最后，对探针的稳定性进行了研究。探针在37 ℃下预孵育1小时，显示发光信号显著降低至1.5%，表明该系统的稳定性有限。使用纯化组分的Fluc PCA将能够快速简便地检测具有高S/B比的PPI，避免伴随组分的影响。虽然该系统可能不适合大规模筛选，由于其有限的稳定性，它可以检测到更大的距离比FRET的相互作用。这将是Fluc PCA在体外的第一次证明，它比其他PPI测定法具有明显的优势。我们的系统能够检测直接PPI，而不会受到复杂细胞环境中PPI介质干扰的风险。
Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system. Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.
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DOI：
10.1093/protein/14.8.529
发表时间：
2001-08-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Arai, R;Ueda, H;Nagamune, T
通讯作者：
Nagamune, T
影响因子：
15
作者：
Magliery, TJ;Wilson, CGM;Regan, L
通讯作者：
Regan, L
影响因子：
11.2
作者：
Paulmurugan, R;Massoud, TF;Gambhir, SS
通讯作者：
Gambhir, SS
DOI：
10.1073/pnas.242594299
发表时间：
2002-11-26
影响因子：
11.1
作者：
Paulmurugan, R;Umezawa, Y;Gambhir, SS
通讯作者：
Gambhir, SS
影响因子：
56.9
作者：
Vassilev, LT;Vu, BT;Liu, EA
通讯作者：
Liu, EA