Controlled Localization of Functionally Active Proteins to Inclusion Bodies Using Leucine Zippers

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（Inclusion bodies, IBs）通常是由聚集蛋白构成的无功能颗粒。然而，部分与类淀粉样肽（amyloid-like peptides）、病毒衣壳蛋白（viral coat proteins）以及纤维素结合结构域（cellulose binding domains, CBDs）融合的蛋白，可形成仍保留细胞内原有功能的包涵体颗粒。本研究尝试构建展示功能性亮氨酸拉链蛋白（leucine zipper proteins, LZs）的CBD包涵体，以作为诱饵定位大肠杆菌（Escherichia coli, E. coli）胞质内的靶蛋白。当以红色荧光蛋白作为靶蛋白进行测试时，显微镜观测结果显示包涵体发出强烈的红色荧光。当以解离常数（dissociation constant, KD）为8~1000 μM的不同亮氨酸拉链蛋白对作为诱饵和猎物蛋白进行测试时，通过荧光成像与流式细胞术观测发现，红色荧光的定位似乎会随亮氨酸拉链蛋白间的亲和力变化而改变。该结果表明，带有亮氨酸拉链标签的CBD包涵体可作为体内基质，在大肠杆菌中捕获胞质蛋白的同时保留其原有活性。此外，可便捷检测蛋白向包涵体的定位，这为大肠杆菌内蛋白质相互作用的工程化改造与分析提供了独特的研究平台。