Controlled Localization of Functionally Active Proteins to Inclusion Bodies Using Leucine Zippers
Controlled Localization of Functionally Active Proteins to Inclusion Bodies Using Leucine Zippers
复制标题
DOI：
10.1371/journal.pone.0097093
复制
发表时间：
2014-06-04
期刊：
影响因子：
3.7
通讯作者：
Lee, Seung-Goo
中科院分区：
文献类型：
--
作者：
Choi, Su-Lim;Lee, Sang Jun;Lee, Seung-Goo
包涵体（IBs）是典型的蛋白质聚集的无功能颗粒。然而，一些蛋白质与淀粉样肽、病毒外壳蛋白和纤维素结合域（CBDs）融合产生的IB颗粒保留了细胞内的原始功能。在这里，我们试图生成显示功能性亮氨酸拉链蛋白（LZs）的CBD IBs作为定位大肠杆菌细胞质蛋白的诱饵。当一种红色荧光蛋白作为靶蛋白进行测试时，显微镜观察显示IBs具有强烈的红色荧光。以K(D)s为8 ~ 1000 μ M的不同LZ对作为诱饵和猎物，通过荧光成像和流式细胞术观察，红色荧光的定位随着LZ之间的亲和力而发生变化。这一结果表明，lz标记的CBD IBs可以作为一种体内基质，在保持其原有活性的同时，在大肠杆菌中捕获胞质蛋白。此外，易于定位到IBs为大肠杆菌中蛋白质-蛋白质相互作用的工程和分析提供了一个独特的平台。
Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with K(D)s of 8-1,000 mu M were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.