Flow cytometric screening of cDNA expression libraries for fluorescent proteins
Flow cytometric screening of cDNA expression libraries for fluorescent proteins
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DOI：
10.1021/bp034308g
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发表时间：
2004-05-01
影响因子：
2.9
通讯作者：
Daugherty, PS
中科院分区：
文献类型：
--
作者：
Bessette, PH;Daugherty, PS
荧光激活细胞分选 (FACS) 用于定量筛选细菌中的 cDNA 表达文库，以寻找编码稀有荧光蛋白的 cDNA。在由 Astrangia lajollaensis 构建的 cDNA 表达文库中，观察到罕见荧光细胞的频率为 200,000 个细菌中的 1 个，通过分选检测、富集和纯化，产生三种不同的绿色荧光蛋白。研究发现，其中两种分离的荧光蛋白的全细胞荧光亮度比广泛使用且已经优化的 EGFP 变体亮 2.5 倍，并且具有新型的含半胱氨酸的发色团。 FACS 在筛选细菌 cDNA 文库方面具有显着优势，因为所需基因可能出现频率较低且具有意想不到的特性。该策略提供了一种高通量、定量的方法，用于从更多样化的生物体中分离荧光蛋白，并且应该可以通过使用可用的荧光指示剂扩展到本质上不发荧光的蛋白质。
Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.