Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.
Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.
复制标题
通过荧光相关光谱监测绿色荧光蛋白及其融合蛋白的体外和体内翻译。
DOI：
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复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
M. Kinjo
中科院分区：
文献类型：
--
作者：
Y. Nomura;H. Tanaka;L. Poellinger;F. Higashino;M. Kinjo
背景
由于蛋白质翻译的过程是一个稀疏分子的事件，因此测量需要高灵敏度。研究分子的候选方法之一是荧光相关光谱法（FCS），它从稀释溶液中波动的荧光信号中收集定量信息。
方法
利用FCS对绿色荧光蛋白（GFP）及其融合蛋白表达质粒的体内外翻译产物进行了检测。
结果
在体外翻译中，产物的数量在质粒浓缩后线性增加90分钟。GFP的自相关函数用扩散时间为0.18 ms的单组分模型拟合，这与从分子量预期的值相同。在GFP标记的缺氧诱导因子-1 α和糖皮质激素受体的情况下，每个拟合结果都用双组分模型显著改善。扩散时间为6 ms的慢组分似乎与核糖体或多核糖体有关。在响应于地塞米松的添加，从胞质溶胶的核转位明显诱导的焦点中的分子数量的减少。
结论
FCS允许监测体外和体内翻译的分子数量、翻译速率和分子量。
BACKGROUND
Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution.
METHODS
Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo.
RESULTS
In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point.
CONCLUSIONS
FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.
影响因子：
13.8
作者：
CUBITT, AB;HEIM, R;TSIEN, RY
通讯作者：
TSIEN, RY
DOI：
10.1073/pnas.95.11.6043
发表时间：
1998-05-26
影响因子：
11.1
作者：
Politz, JC;Browne, ES;Pederson, T
通讯作者：
Pederson, T
DOI：
10.1073/pnas.95.12.6803
发表时间：
1998-06-09
影响因子：
11.1
作者：
Llopis, J;McCaffery, JM;Tsien, RY
通讯作者：
Tsien, RY