Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening
Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening
复制标题
基质工程实现荧光液滴捕获，用于基于 IVC-FACS 的超高通量筛选
DOI：
10.1021/acs.analchem.6b01712
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发表时间：
2016-09-06
影响因子：
7.4
通讯作者：
Yang, Guang-Yu
中科院分区：
文献类型：
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作者：
Ma, Fuqiang;Fischer, Michael;Yang, Guang-Yu
基于体外区室化的荧光激活细胞分选（IVC-FACS）是酶定向进化的强大筛选工具。然而，IVC-FACS 的效率受到荧光报道分子从液滴中扩散出来的趋势的限制，这使目标基因的基因型和表型脱钩。在这里，我们提出了一种称为荧光液滴捕获（FDE）的新策略来解决这个问题。底物的极性设计使其能够穿过油相，与酶反应并产生不透油的荧光产物，该产物保留在液滴内。使用两种不同的底物工程策略设计了几种 FDE 底物，用于检测磷酸三酯酶、羧酸酯酶和糖苷酶活性。从大量过量的非活性细胞中富集稀有磷酸三酯酶活性细胞的模型筛选测定表明，使用 FDE 底物可实现的富集效率高达 900 倍：此类 IVC-FACS 系统中迄今为止报道的最高效率。因此，FDE 提供了一种严格控制酶促反应开始、最小化液滴串扰并降低背景荧光的方法。因此，它可以作为 IVC-FACS 筛选酶、抗体和其他蛋白质的有用策略。
In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.