一种用于体内磷酸盐追踪的新型分析方法。
A novel analytical method for in vivo phosphate tracking.
作者信息
Gu Hong, Lalonde Sylvie, Okumoto Sakiko, Looger Loren L, Scharff-Poulsen Anne Marie, Grossman Arthur R, Kossmann Jens, Jakobsen Iver, Frommer Wolf B
机构信息
Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA.
出版信息
FEBS Lett. 2006 Oct 30;580(25):5885-93. doi: 10.1016/j.febslet.2006.09.048. Epub 2006 Oct 2.
Abstract
Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.
摘要
通过将预测的集胞藻磷酸结合蛋白（PiBP）与eCFP和Venus融合，构建了用于磷酸盐（P(i)）的基因编码荧光共振能量转移（FRET）传感器（FLIPPi）。纯化的无机磷酸盐荧光指示剂蛋白（FLIPPi）中，荧光团连接在同一PiBP叶上，其FRET效率呈现出P(i)依赖性增加。FLIPPi亲和力突变体涵盖了八个数量级的P(i)变化。共表达低亲和力FLIPPi和Na(+)/P(i)共转运体的COS-7细胞在灌注100 microM P(i)时表现出FRET变化，证明了PiT2对P(i)的浓缩摄取。FLIPPi传感器适用于实时监测活细胞中的P(i)代谢，为通量组学、病理生理学分析或细胞迁移过程中P(i)的变化提供了一种新工具。
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