A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC)
A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC)
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DOI：
10.2144/000113941
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发表时间：
2012-11-01
期刊：
影响因子：
2.7
通讯作者：
Blatt, Michael R.
中科院分区：
文献类型：
--
作者：
Grefen, Christopher;Blatt, Michael R.
基因表达和二元相互作用技术是塑造我们对蛋白质复合物理解的重要工具。然而，当前大多数蛋白-蛋白相互作用技术的一个固有缺陷是，当使用几个单独的质粒时，融合蛋白的表达水平存在可变性。在这里，我们描述了一种新的基于重组的克隆策略，称为2in1，它可以在单个质粒的基础上逐个细胞地共表达融合蛋白。我们通过开发比例双分子荧光互补试验（rBiFC）证明了2in1的效用，其中两个候选基因同时克隆到一个包含内部荧光标记的单一载体骨架中，用于表达控制和比例分析。rBiFC显著提高了蛋白质相互作用结果的可信度，允许不同蛋白质对之间的比率比较。除了用于rBiFC外，2in1还可以很容易地引入其他依赖多基因表达的载体系统，并在未来的合成生物学方法中被证明是可行的。
Gene expression and binary interaction techniques are vital tools that shape our understanding of protein complexes. An inherent flaw, however, with most current protein-protein interaction techniques is the variability in expression levels for fusion proteins when using several individual plasmids. Here, we describe a novel recombination-based cloning strategy called 2in1 that enables co-expression of fusion proteins on a cell-by-cell basis from a single plasmid. We demonstrate the utility of 2in1 through the development of a ratiometric bimolecular fluorescence complementation assay (rBiFC), in which both candidate genes are simultaneously cloned into a single vector backbone containing an internal fluorescent marker for expression control and ratiometric analysis. rBiFC significantly increases the credibility of protein-protein interaction results allowing ratiometric comparison between different protein pairs. In addition to its use in rBiFC, 2in1 can be introduced easily into other vector systems that rely on multiple gene expression and prove feasible in future synthetic biological approaches.