FAST 蛋白报告基因系统,依据巴黎高等师范学院和巴黎文理研究大学居里研究所专利技术研发生产,目前已经得到广泛应用。 FAST 蛋白报告基因系统是一种革命性的双组分荧光标记系统,只有当 FAST 蛋白与荧光底物特异性的结合后,复合物才能被激发产生荧光,实现活细胞中对目标蛋白的标记。FAST 蛋白与荧光底物动态可逆的结合方式,给予 FAST 蛋白报告基因系统独特的可逆标记特性:在细胞生长体系中添加或洗出荧光底物,即可打开或关闭细胞内含 FAST 标签目标蛋白的荧光,完全实现按需显示。FAST 蛋白报告基因系统是一种全新的技术方案,可用于超分辨率成像,生物传感器设计,顺序多重成像,同样也为基因组编辑和生物生产等提供了不一样的荧光标记选项。FAST 蛋白只有 14KD 大小,与目的蛋白融合表达时,不影响目的蛋白的活性和构象,保证目的蛋白在细胞中原始的功能。与传统 GFP, mCherry,Halo,SNAP-tag 等报告基因系统相比,FAST 蛋白报告基因系统有独特的优势,如图所示: 应用 1:厌氧条件下荧光检测 美国特拉华大学 Terry Papoutsakis 教授利用 FAST 标记体系(应用了 TFLime 与 TFCoral 荧光配体)实现了在厌氧培养条件下丙酮丁醇梭菌 (Clostridium acetobutylicum) 的高效荧光标记。以此为基础,该团队建立了基于流式细胞仪和酶标仪的丙酮丁醇梭菌蛋白荧光监测系统,并分析了一系列蛋白的表达量或是蛋白定位。A strongly fluorescing anaerobic reporter and protein-tagging system for Clostridium organisms based on the Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein. Appl. Environ. Microbiol. AEM-00622 (2019).
FAST and HMBR (TFLime) are an efficient system to view protein localization within C. acetobutylicum cells. Shown is C. acetobutylicum ZapA-FAST with 20 μM HMBR (left), brightfield (middle), and merge (right) using confocal microscopy. Each row represents a different field of view.
应用 2:生物膜研究 法国索邦大学 Nelly Henry 博士的研究表明,FAST 标记体系可对细菌生物膜中的生物群落功能进行实时、定量分析。通过比较常规荧光蛋白(GFP 或 mCherry)与 FAST 标记,她的研究小组发现由于细菌培养过程中氧气浓度降低,GFP 或 mCherry 荧光信号在生物膜生长数小时后(2-5 小时)即达到峰值,不能如实反映生物膜生长状况;而 FAST 标记体系则不受氧浓度影响,能够实时标记生物膜,且荧光强度高于常规荧光蛋白两个数量级。FAST 标记体系在研究生物膜形成的复杂环境中有十分明显的优越性。The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics. Sci. Rep. 8(1), 10336 (2018) 应用 3:蛋白相互作用研究 SplitFAST 报告基因系统,是利用 FAST 蛋白特点进行的改造而成。原理是将 FAST 蛋白分拆为两个独立原件:FAST 蛋白 N 端 (N-FAST ) 和 FAST 蛋白 C 端 (C-FAST),将可能有相互作用目的蛋白分别与两个独立原件融合表达。如图所示:
Figure. Use of splitFAST for imaging the evolution of MEK1/ERK2 interaction upon epidermal growth factor (EGF) stimulation. HMBR-labeled HeLa cells co-expressing MEK1-NFAST and mCherry-ERK2-CFAST10 were imaged after stimulation with EGF
近 2 年发表文献
· Nat. Commun. 2021 – Lu, Z., Peng, B., Ebert, B. E., Dumsday, G., & Vickers, C. E. Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast.
· In Review 2021 – Flaiz, M., Ludwig, G., Bengelsdorf, F. R., & Dürre, P. Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum.
· bioRxiv 2020 – Mineev, K. S., Goncharuk, S. A., Goncharuk, M. V., Povarova, N. V., Baleeva, N. S., Smirnov, A. Y., … & Baranov, M. S. NanoFAST: Structure-based design of a small fluorogen-activating protein with only 98 amino acids.
· Chem. Eur. J. 2020 – Myasnyanko, I. N., Gavrikov, A. S., Zaitseva, S. O., Smirnov, A. Y., Zaitseva, E. R., Sokolov, A. I., … & Baranov, M. S. Color tuning of fluorogens for FAST fluorogen‐activating protein.
Sci. Rep. 2020 – Chekli, Y., Peron-Cane, C., Dell’Arciprete, D., Allemand, J. F., Li, C., Ghigo, J. M., … & Beloin, C. Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST.
· PLoS Pathog. 2020 – Peron-Cane, C., Fernandez, J. C., Leblanc, J., Wingertsmann, L., Gautier, A., Desprat, N., & Lebreton, A. Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.
· Mbio 2020 – Charubin, K., Modla, S., Caplan, J. L., & Papoutsakis, E. T. Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture.
· Appl. Environ. Microbiol. 2020 – Charubin, K., Streett, H., & Papoutsakis, E. T. Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins.
· Appl. Environ. Microbiol. 2019 – Streett, H. E., Kalis, K. M., & Papoutsakis, E. T. A strongly fluorescing anaerobic reporter and protein-tagging system for Clostridium organisms based on the fluorescence-activating and absorption-shifting tag protein (FAST).
· Chem. Eur. J. 2019 – Povarova, N. V., Zaitseva, S. O., Baleeva, N. S., Smirnov, A. Y., Myasnyanko, I. N., Zagudaylova, M. B., … & Mishin, A. S. Red‐shifted substrates for FAST fluorogen‐activating protein based on the GFP‐like chromophores.
· ACS Chem. Biol. 2019 – Smith, E. M., Gautier, A., & Puchner, E. M. Single-molecule localization microscopy with the fluorescence-activating and absorption-shifting tag (FAST) system.
· Nanoscale 2019 – Venkatachalapathy, M., Belapurkar, V., Jose, M., Gautier, A., & Nair, D. Live cell super resolution imaging by radial fluctuations using fluorogen binding tags.
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