Sadoine Mayuri, Castro-Rodríguez Vanessa, Poloczek Tobias, Javot Helene, Sunal Erdem, Wudick Michael M, Frommer Wolf B
Heinrich-Heine-University, Institute of Molecular Physiology, Düsseldorf, Germany.
Aix Marseille Univ, CEA, CNRS, BIAM, Saint Paul-Lez-Durance, France.
Bio Protoc. 2020 Oct 5;10(19):e3773. doi: 10.21769/BioProtoc.3773.
Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites . Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating data. This protocol provides a robust methodology for binding assays of newly designed sensors. Here we present a detailed protocol for purification and characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.
基因编码生物传感器是用于离子和代谢物定量可视化的强大工具。此类生物传感器的设计和优化通常需要对大量变体进行分析。所确定的传感器特性，如底物特异性、亲和力、响应范围、动态范围和信噪比，对于评估数据很重要。本方案提供了一种用于新设计传感器结合测定的可靠方法。在这里，我们展示了一个用于基因编码传感器纯化和表征的详细方案，以His亲和标签的GO-（绿-橙）套娃CaMP6s钙传感器为例。GO-套娃传感器基于在目标结合蛋白内单步插入一个包含两个嵌套荧光蛋白的盒式结构，即环状排列的荧光绿色FP（cpGFP）和大斯托克斯位移LSSmOrange，从而产生利用cpGFP荧光中分析物触发变化的比率传感器。
