LEC-BiFC: a new method for rapid assay of protein interaction
LEC-BiFC: a new method for rapid assay of protein interaction
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LEC-BiFC：一种快速测定蛋白质相互作用的新方法
DOI：
10.3109/10520295.2010.483068
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发表时间：
2011-08-01
影响因子：
1.6
通讯作者：
Xia, B.
中科院分区：
文献类型：
--
作者：
Lin, J.;Wang, N.;Xia, B.
蛋白质-蛋白质相互作用在大多数生物过程中起着重要作用。双分子荧光互补技术（BiFC）以其简单、直观的特点成为研究细胞内蛋白质相互作用的一种新方法。然而，这种方法受到蛋白质之间没有特异性相互作用的背景荧光的限制。我们在这里报告了一个Venus BiFC片段中的点突变（V150 L），该突变有效地降低了BiFC测定的背景荧光。此外，通过将该修饰的BiFC与线性表达盒（LEC）相结合，我们开发了一种简单快速的蛋白质相互作用分析方法（LEC-BiFC），并以Bcl-X（L）与巴克BH 3肽相互作用为例进行了验证。整个分析过程可以在两天内完成，筛选出数十个突变体。LEC-BiFC可以很容易地应用于任何配备荧光显微镜的实验室。
Protein-protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein-protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC-BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl-X(L) and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC-BiFC can be applied easily in any lab equipped with a fluorescence microscope.