Deo S K, Daunert S
Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
Fresenius J Anal Chem. 2001 Feb;369(3-4):258-66. doi: 10.1007/s002160000646.
Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.
通过基因组学生成大量药物靶点以及通过组合化学构建大型化合物库方面的最新进展，推动了利用新的高通量筛选（HTS）方法在药物发现领域取得进展。自动化和HTS技术在临床诊断等领域也备受青睐。基于发光的检测方法已成为HTS中基于放射性标记检测方法的替代方法，因为它们在具备易于操作特点的同时接近放射性检测的灵敏度，这使得它们易于小型化。发光蛋白具有降低试剂和操作成本的优势，因为可以通过使用基因工程工具大量生产。在这方面，来自维多利亚多管水母的两种天然存在且经重组产生的发光蛋白，即水母发光蛋白和绿色荧光蛋白（GFP），在不同研究领域的众多分析应用中引起了关注。水母发光蛋白天然具有生物发光特性，因此几乎没有相关背景信号，这使其能够检测到阿托摩尔水平。鉴于GFP是一种自发荧光蛋白，荧光发射无需添加任何辅助因子，它已成为各种应用中的首选报告基因。此外，具有不同发光和光谱特性的各种GFP突变体的产生激发了人们对这种蛋白质的更多兴趣。在本综述中，我们重点关注水母发光蛋白和GFP在开发高灵敏度检测方法中的应用，这些方法在药物发现和高通量分析中具有应用价值。
