Noncytotoxic DsRed derivatives for whole-cell labeling.
Noncytotoxic DsRed derivatives for whole-cell labeling.
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DOI：
10.1007/978-1-61737-950-5_17
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Glick, Benjamin S
中科院分区：
文献类型：
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作者：
Strack, Rita L;Keenan, Robert J;Glick, Benjamin S
荧光蛋白(FP)是生物医学研究的宝贵工具。有用的FP具有理想的荧光特性，如亮度和光稳定性，但局限性是许多橙色、红色和远红FP在胞浆中表达时具有细胞毒性。这种细胞毒性源于聚集。为了减少聚集，我们在DsRed-Express的表面进行了工程，以生成DsRed-Express2，这是一种在细菌和哺乳动物细胞中无细胞毒性的高度溶解的四聚体FP。DsRed-Express2的定向进化产生了颜色变体E2-橙色、E2-红/绿色和E2-深红色。这些变体可以用来标记整个细胞，用于使用显微镜或流式细胞术进行单色和多色实验。描述了减少低聚FP的高阶聚集和分析FP在大肠杆菌和HeLa细胞中的细胞毒性的方法。
Fluorescent proteins (FPs) are invaluable tools for biomedical research. Useful FPs have desirable fluorescence properties such as brightness and photostability, but a limitation is that many orange, red, and far-red FPs are cytotoxic when expressed in the cytosol. This cytotoxicity stems from aggregation. To reduce aggregation, we engineered the surface of DsRed-Express to generate DsRed-Express2, a highly soluble tetrameric FP that is noncytotoxic in bacterial and mammalian cells. Directed evolution of DsRed-Express2 yielded the color variants E2-Orange, E2-Red/Green, and E2-Crimson. These variants can be used to label whole cells for single- and multi-color experiments employing microscopy or flow cytometry. Methods are described for reducing the higher-order aggregation of oligomeric FPs and for analyzing FP cytotoxicity in E. coli and HeLa cells.