trap:一种基于trap的活细胞蛋白翻译监测技术

N. Griesche, V. Pesch, Rohit Nalavade, S. Weber, Ireen König, M. Schölling, Christoph Möhl, Sybille Krau
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引用次数: 1

摘要

传统上,蛋白质翻译的研究依赖于系统,在系统中,细胞已经被裂解,预先确定感兴趣的蛋白质水平。然而,这些检测并不能实时反映活细胞中的蛋白质合成,而是分析给定孵育时间后的蛋白质水平,导致基于实验参数的结果存在局限性。为了克服这个问题,我们之前建立了一种基于光漂白后荧光恢复(FRAP)的技术来监测活细胞中的蛋白质翻译。为此,将感兴趣的蛋白融合到绿色荧光蛋白(GFP)中,在细胞系中表达。在漂白整个细胞后,感兴趣的蛋白的荧光信号丢失,允许捕获新翻译的GFPtagged蛋白随时间的信号恢复。在这里,我们提出了使用不同荧光染料的该技术的两个改进版本:tFRAP(翻译FRAP)。对于trap的第一个改进版本,我们将第二种荧光染料,红色荧光蛋白(RFP)插入到相同的表达载体中,该表达载体驱动目标蛋白的表达,融合到由第二个启动子驱动的GFP中。对于tFRAP的第二个改进版本,我们将感兴趣的蛋白质融合到光切换染料Dendra2中。这两种改进版本都允许针对不同转染率的单个细胞校正感兴趣蛋白的荧光信号强度。这两种先进的技术是量化活细胞翻译速率的有力工具,对未来mRNA翻译的研究具有重要意义。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
tFRAP: A FRAP-Based Technique to Monitor Protein Translation in Living Cells
Traditionally, studies on protein translation rely on systems, in which cells have been lysed prior determination of levels of the protein of interest. However, these assays do not reflect the protein synthesis in living cells in real time, but analyze protein levels after a given incubation time, leading to limitations in results based on experimental parameters. To overcome this problem, we have previously established a Fluorescence recovery after photobleaching (FRAP)-based technique to monitor protein translation in living cells. For this, the protein of interest fused to green fluorescent protein (GFP) is expressed in cell lines. After bleaching the entire cell, the fluorescent signal of the protein of interest is lost, allowing to capture the signal recovery of newly translated GFPtagged protein over time. Here we present two improved versions of this technique using different fluorescent dyes: tFRAP (translational FRAP). For the first improved version of tFRAP we have inserted a second fluorescent dye, red fluorescent protein (RFP), into the same expression vector that drives expression of the protein of interest fused to GFP driven by a second promoter. For the second improved version of tFRAP we have fused our protein of interest to a photo-switchable dye, Dendra2. Both improved versions allow to correct the fluorescence signal intensity of the protein of interest for different transfection rates of individual cells. These two advanced techniques are new powerful tools for quantifying translation rates in living cells and will be useful in future studies on mRNA translation.
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