A high-throughput in vivo selection method for luciferase variants
A high-throughput in vivo selection method for luciferase variants
复制标题
荧光素酶变体的高通量体内选择方法
DOI：
10.1016/j.snb.2018.06.047
复制
发表时间：
2018-11-10
影响因子：
8.4
通讯作者：
Tang, Zhuo
中科院分区：
文献类型：
--
作者：
Sun, Meiling;Fu, Zhicong;Tang, Zhuo
荧光素酶催化其特殊底物并在氧化反应过程中发光，被广泛用作监测基因表达、蛋白-蛋白相互作用和肿瘤对治疗反应的光学传感器。然而，由于光信号衰减快、稳定性低、发光强度差等缺点，使得光信号在样品检测中难以被捕获。为了获得具有改进特征的荧光素酶变体，传统的方法通常是基于CCD成像技术或群体采摘，这些方法的通量低，耗时且费力。在此，我们描述了一种新的荧光素酶筛选方法，称为快速文库收缩法（FLSM），该方法可以有效地从庞大而复杂的变体库中选择所需的突变体，而无需逐个分析突变体。基于FLSM，我们成功地获得了一个名为M43IL119Q的Gluc变体，该变体的发光强度增加了3倍，并且具有稳定的发光动力学。该选择方法易于使用且具有成本效益，因此使其成为荧光素酶快速进化的有前途的平台。
Luciferase catalyzes its special substrate and light emits during the oxidation reaction, which is widely used as an optical sensor to monitor gene expression, protein-protein interaction and tumor response to therapy. However, limitations like the rapid decay, low stability and poor intensity of light emission make the optical signal hard to be captured in the sample detection. To obtain luciferase variants with improved features, traditional methods are usually based on CCD imaging technology or colony picking, which are low-throughput, time and labor consuming. Herein, we describe a novel luciferase screening method called fast library shrink method (FLSM), which could efficiently help select the desired mutant from the huge and complicated pool without analyzing the variants one by one. Based on FLSM, we successfully obtained one Gluc variant named M43IL119Q, which shows a 3-fold increase in the intensity of light emission as well as a stable light emission kinetics. The selection method is ready-to-use and cost-effective, thus making it a promising platform for the fast evolution of luciferases.