An efficient method for FITC labelling of proteins using tandem affinity purification
An efficient method for FITC labelling of proteins using tandem affinity purification
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DOI：
10.1042/bsr20181764
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发表时间：
2018-12-21
影响因子：
4
通讯作者：
Bose, Kakoli
中科院分区：
文献类型：
--
作者：
Chaganti, Lalith K.;Venkatakrishnan, Navneet;Bose, Kakoli
基于荧光的分析是研究构象变化、酶动力学、动力学和分子相互作用的极其多样化、敏感和强大的实验方法。大多数这些实验方法的先决条件是用一个或多个具有所需光物理性质的外部荧光团标记感兴趣的蛋白质。荧光素异硫氰酸酯（荧光素异硫氰酸酯，FITC）由于其高量子效率和共轭稳定性，是这类实验方法中使用最广泛的荧光标记试剂。然而，这种标记反应的瓶颈是对高蛋白质浓度的要求，在标记过程中保持蛋白质的稳定性，以及在荧光研究之前，由于没有有效去除未反应的FITC而导致的高背景荧光。因此，为了克服这些不足或局限性，我们修改了现有的方案，在靶蛋白的N端和c端引入串联亲和纯化标签。利用这种改进的方法，我们有效地标记了目标蛋白，显著减少了未反应的FITC的沉淀、降解和背景荧光。这种简便快速的技术也可用作其他荧光团标记程序的基础，因此在光谱学研究中具有广泛的应用。
Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.