Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications
Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications
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DOI：
10.1016/j.ab.2005.07.015
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发表时间：
2005-10-01
影响因子：
2.9
通讯作者：
Roda, A
中科院分区：
文献类型：
--
作者：
Branchini, BR;Southworth, TL;Roda, A
来自北美萤火虫Photinus pyralis的光发射，其发射黄绿色（557-nm）光，被广泛认为是已知的最有效的生物发光系统，使得这种荧光素酶成为监测基因表达的极好工具。在这里，我们目前的研究，导致生产一组红色和绿色发光的荧光素酶突变体的生物发光特性适合扩大使用的P. pyralis系统的双色报告分析，生物传感器测量与内部控制，和成像技术。使用诱变方法的组合，我们确定Ser 284 Thr突变足以产生具有615 nm的生物发光最大值、窄的发射带宽和有利的动力学性质的优异的发红光荧光素酶。此外，我们开发了一种荧光素酶，含有变化Va 124 Ile，Gly 246 Ala和Phe 250 Ser，其发射最大值蓝移到549 nm，提供了一组酶，其生物发光最大值由66 nm分离。模型研究表明，在使用一组光学滤波器的测定中，可以在阿托摩尔水平和更高的7个数量级上检测到酶。此外，在作为对照的Ser 284 Thr酶存在下，可以在10，000倍范围内测量绿色光发射。本文中给出的P. pyralis突变体的结果提供了证据，证明使用这些仅需要单一底物的新型蛋白质进行同时多分析物测定开发是可行的。(C)2005年爱思唯尔公司All rights reserved.
Light emission from the North American firefly Photinus pyralis, which emits yellow green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Va124Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate. (C) 2005 Elsevier Inc. All rights reserved.