Choi Su-Lim, Lee Sang Jun, Yeom Soo-Jin, Kim Hyun Ju, Rhee Young Ha, Jung Heung-Chae, Lee Seung-Goo
Biochemicals and Synthetic Biology Research Center, KRIBB, Yuseong-gu, Daejeon, Korea; Department of Bioscience and Biotechnology, Chungnam National University, Yuseong-gu, Daejeon, Korea.
Infection and Immunity Biology Research Center, KRIBB, Yuseong-gu, Daejeon, Korea; Korea University of Science and Technology, Yuseong-gu, Daejeon, Korea.
PLoS One. 2014 Jun 4;9(6):e97093. doi: 10.1371/journal.pone.0097093. eCollection 2014.
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 KDs of 8-1,000 µM 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.
包涵体（IBs）通常是聚集蛋白的无功能颗粒。然而，一些与淀粉样肽、病毒衣壳蛋白和纤维素结合结构域（CBDs）融合的蛋白会产生在细胞中保留原始功能的IB颗粒。在这里，我们试图生成展示功能性亮氨酸拉链蛋白（LZs）的CBD包涵体，作为在大肠杆菌中定位胞质蛋白的诱饵。当测试红色荧光蛋白作为靶蛋白时，显微镜观察表明包涵体发出强烈的红色荧光。当测试不同解离常数（KDs）为8 - 1000 μM的LZ对作为诱饵和猎物时，通过荧光成像和流式细胞术观察到，红色荧光的定位似乎随着LZ之间的亲和力而变化。该结果表明，带有LZ标签的CBD包涵体可作为体内基质，在保持其原始活性的同时捕获大肠杆菌中的胞质蛋白。此外，易于检测到对包涵体的定位为大肠杆菌中蛋白质 - 蛋白质相互作用的工程设计和分析提供了一个独特的平台。
