Creation of Artificial Luciferases for Bioassays
Creation of Artificial Luciferases for Bioassays
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DOI：
10.1021/bc400411h
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发表时间：
2013-12-01
影响因子：
4.7
通讯作者：
Tao, Hiroaki
中科院分区：
文献类型：
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作者：
Kim, Sung Bae;Torimura, Masaki;Tao, Hiroaki
本研究展示了用于生物测定的人工酶（ALuc）的创建，灵感来自桡足类生物酶的序列比对。从比对中提取共有氨基酸使我们能够产生一系列具有独特光学性质和序列身份的ALucs，这些性质和序列身份明显不同于任何现有的桡足类荧光素酶。例如，一些ALucs表现出热稳定性、显著延长的光学强度、宽的半峰全宽和强的光学强度。在不同的生物测定，包括哺乳动物的双杂交测定，活细胞成像，单链探针，生物发光胶囊，和生物发光抗体的实际适用性的酶作为光学读出检查。我们通过生物信息学分析进一步确定ALucs的物理性质，最后讨论ALucs独特性质的详细问题。本研究展示了如何创建具有优异光学性能的生物测定人工酶，并鼓励研究人员制造自己独特的人工酶与设计的性能和功能。
The present study demonstrates the creation of artificial luciferases (ALuc) for bioassays, inspired by a sequence alignment of copepod luciferases. Extraction of the consensus amino acids from the alignment enabled us to generate a series of ALucs with unique optical properties and sequential identities that are clearly different from those of any existing copepod luciferase. For example, some ALucs exhibited heat stability, dramatically prolonged optical intensities, broad full width at half-maximum, and strong optical intensities. The practical suitability of the luciferases as an optical readout was examined in diverse bioassays, including mammalian two-hybrid assays, live cell imaging, single-chain probes, bioluminescent capsules, and bioluminescent antibodies. We further determine the physical properties of ALucs through bioinformatic analysis and finally discuss detailed issues on the unique properties of ALucs. The present study shows how to create the artificial enzymes with excellent optical properties for bioassays and encourages researchers to fabricate their own unique artificial enzymes with designed properties and functionalities.