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首页> 外文会议>Conference on Laser Microscopy, Jul 7-8, 2000, Amsterdam, Netherlands >Multi-pixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation
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Multi-pixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

机译：COS-7细胞中绿色荧光蛋白（GFP）的多像素光谱成像：折叠动力学和生色团形成

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摘要

Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hrs for gene expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hrs, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both to the typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hrs, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multipixel spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.
机译：在活COS-7肾细胞中表达的绿色荧光蛋白（GFP）的光谱分辨成像可区分亚细胞定位，并区分了蛋白折叠和生色团的形成过程。用质粒pEGFP-N1质粒转染COS-7肾细胞，然后温育15小时以进行基因表达。在不同的时间间隔，通过荧光显微镜检查细胞并通过光谱成像系统进行分析。 7小时后，在细胞质中检测到GFP，以局部形式浓缩。初始GFP的光谱显示出几种成分，这些成分既属于典型的荧光信号，也属于非特异性的指纹。在10小时和15小时时，发现GFP在细胞质和细胞核中扩散，并且GFP的特定光谱在稍后时间占主导。典型的GFP光谱指纹图谱是内部氧化反应后蛋白质折叠和生色团形成的结果。这种折叠和生色团形成过程，直至最终构象，是通过光谱成像检测到的，位于细胞核而不是细胞质中。因此，荧光显微镜结合多像素光谱成像的方法证明了导致GFP构象的不同生化途径。

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