Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.
Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.
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DOI：
10.1371/journal.pone.0064597
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Kirimura K
中科院分区：
文献类型：
--
作者：
Honda Y;Kirimura K
由于柠檬酸盐是活细胞中起重要作用的关键代谢中间体，因此柠檬酸盐的指示剂，特别是那些适用于其体内检测和定量的指示剂，在生物化学研究和工业应用中都引起了极大的兴趣。我们通过融合循环排列荧光蛋白（cpFP）和细菌组氨酸激酶CitA的周质结构域（其可以以高特异性结合柠檬酸盐）来产生柠檬酸盐的新型荧光指示剂。使用其中一种基于cpFP的指示剂（名为CF 98）观察到比率荧光信号变化：加入柠檬酸盐后，504 nm处的激发峰与413 nm处的峰降低成比例增加，适用于结合化合物的内置定量估计。我们证实，CF 98可用于检测柠檬酸盐在体外的毫摩尔水平在0.1至50 mM的范围内，具有高选择性;即使在其他有机酸，如异柠檬酸和苹果酸的存在下，CF 98的荧光强度保持不受影响。我们最终证明了CF 98在体内的适用性，以估计在大肠杆菌中的细胞内柠檬酸盐浓度共表达的基因编码CF 98和柠檬酸盐载体CitT。新的指示剂CF 98可以是一种特异性和简单的检测工具，柠檬酸盐在体外和一个非侵入性的工具，用于实时估计细胞内的化合物在体内的浓度。
Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estimation of the intracellular citrate concentration in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estimation of intracellular concentrations of the compound in vivo.
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影响因子：
1.6
作者：
Maruyama, J;Nakajima, H;Kitamoto, K
通讯作者：
Kitamoto, K
影响因子：
2.7
作者：
Fehr, M;Lalonde, S;Frommer, WB
通讯作者：
Frommer, WB
影响因子：
3.6
作者：
BOTT, M;MEYER, M;DIMROTH, P
通讯作者：
DIMROTH, P
DOI：
10.1073/pnas.142089199
发表时间：
2002-07-23
影响因子：
11.1
作者：
Fehr, M;Frommer, WB;Lalonde, S
通讯作者：
Lalonde, S
影响因子：
2.8
作者：
Kirimura, Kohtaro;Ogawa, Satoshi;Kino, Kuniki
通讯作者：
Kino, Kuniki