A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range

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• 作者：Lin Xue ; Efthymia Prifti ; Kai Johnsson
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：April 27, 2016
• 年：2016
• 卷：138
• 期：16
• 页码：5258-5261
• 全文大小：311K
• 年卷期：0
• ISSN：1520-5126

文摘

We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.