Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
PLoS One. 2013;8(1):e55049. doi: 10.1371/journal.pone.0055049. Epub 2013 Jan 22.
We have constructed a set of plasmids that allow efficient expression of both N- and C-terminal fusions of proteins of interest to fluorescent proteins mCherry, Citrine, CFP and GFP in the Gram-positive pathogen Streptococcus pneumoniae. In order to improve expression of the fluorescent fusions to levels that allow their detection by fluorescence microscopy, we have introduced a 10 amino acid tag, named i-tag, at the N-terminal end of the fluorescent proteins. This caused increased expression due to improved translation efficiency and did not interfere with the protein localization in pneumococcal bacteria. Localizing fluorescent derivatives of FtsZ, Wzd and Wze in dividing bacteria validated the developed tools. The availability of the new plasmids described in this work should greatly facilitate studies of protein localization in an important clinical pathogen.
我们构建了一组质粒，可在革兰氏阳性病原体肺炎链球菌中有效表达目的蛋白的 N 端和 C 端融合物与荧光蛋白 mCherry、Citrine、CFP 和 GFP。为了提高荧光融合物的表达水平，使其能够通过荧光显微镜检测，我们在荧光蛋白的 N 端引入了一个名为 i-tag 的 10 个氨基酸标签。这导致表达增加，因为翻译效率提高，并且不会干扰肺炎球菌中蛋白质的定位。在分裂细菌中定位 FtsZ、Wzd 和 Wze 的荧光衍生物验证了所开发工具的有效性。本工作中描述的新质粒的可用性应大大促进对重要临床病原体中蛋白质定位的研究。
