Selective dye-labeling of newly synthesized proteins in bacterial cells
Selective dye-labeling of newly synthesized proteins in bacterial cells
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DOI：
10.1021/ja054643w
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发表时间：
2005-10-19
影响因子：
15
通讯作者：
Tirrell, DA
中科院分区：
文献类型：
--
作者：
Beatty, KE;Xie, F;Tirrell, DA
本文报道了利用Cu（I）催化的炔基氨基酸侧链与荧光染料3-叠氮基-7-羟基香豆素之间的环加成反应对大肠杆菌细胞中新合成的蛋白质进行荧光标记。该方法涉及通过非天然氨基酸高炔丙基甘氨酸（Hpg）或乙炔基苯丙氨酸（Eth）共翻译标记蛋白质，然后用染料处理。作为证明，表达模型蛋白芽孢杆菌RNA酶抑制剂并用Cu（I）和3-叠氮基-7-羟基香豆素处理过夜。通过共聚焦显微镜检查处理的细胞显示，仅观察到用Cu（I）和活性染料处理的炔基-芽孢杆菌RNA酶抑制剂的强荧光增强。细胞荧光呈点状，凝胶电泳证实标记的芽孢杆菌RNA酶抑制剂定位于包涵体。其他蛋白质几乎没有荧光。通过荧光测定法对处理过的细胞进行的检查表明，添加有Eth或Hpg的培养物在标记后显示出8至14倍的荧光强度增强。添加蛋白质合成抑制剂将发射强度降低至略高于背景的水平，证实了细菌细胞中新合成蛋白质的选择性标记。
We describe fluorescence labeling of newly synthesized proteins inEscherichia colicells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.