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首页> 外文期刊>BMC Biotechnology >Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments
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Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments

机译：演示了使用纯化的萤火虫荧光素酶片段进行的蛋白质片段互补测定

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Background 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 F?rster 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. Results 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. Conclusion 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.
机译：背景技术人类交互组预计包含150,000至300,000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以及酯共振能量转移（FRET）的最广泛使用的方法之一。迄今为止，由于萤火虫荧光素酶（Fluc）的PCA具有高灵敏度，高信噪比（S / B）和高灵敏度，因此已在体内，培养细胞和无细胞裂解物中成功应用了基于萤火虫荧光素酶（Fluc）的PCA。可逆反应。在这里，我们显示该测定法还可以用于具有出乎意料的快速动力学的纯化蛋白质。结果制备了均与雷帕霉素依赖性相互作用蛋白对融合的分裂的Fluc片段，并在大肠杆菌系统中表达，并纯化至同质。当这些蛋白质用于PCA来检测雷帕霉素依赖性PPI时，它们能够以高S / B比快速检测（〜1？s）PPI。当显示可废除基于GFP突变体的FRET的Fn7-8结构域（长度为7？nm）插入到分裂的Fluc和FKBP12之间作为刚性连接子时，它仍显示出一定的响应，表明对相互作用对象大小的限制较少。最后，研究了探针的稳定性。探针在37摄氏度下预孵育1？h时，发光信号显着降低至1.5％，表明该系统的稳定性有限。结论使用纯化组分的Fluc PCA将能够快速，方便地检测具有高S / B比的PPI，避免了伴随组分的影响。尽管由于稳定性有限，该系统可能不适合大规模筛选，但它可以检测到比FRET更大的距离上的相互作用。这将是Fluc PCA在体外的首次展示，它比其他PPI分析具有明显的优势。我们的系统能够检测直接的PPI，而不会受到复杂细胞环境中PPI介体干扰的风险。

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