Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST) , 16-1 Onogawa, Tsukuba 305-8569, Japan.
Bioconjug Chem. 2013 Dec 18;24(12):2067-75. doi: 10.1021/bc400411h. Epub 2013 Nov 27.
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.
本研究通过对桡足类荧光素酶的序列比对，设计并合成了一系列具有独特光学性质和序列同一性的人工荧光素酶（ALuc），用于生物检测。从比对中提取的共识氨基酸使我们能够生成一系列具有独特光学性质和序列同一性的 ALuc，与任何现有的桡足类荧光素酶明显不同。例如，一些 ALuc 表现出热稳定性、光学强度显著延长、全宽半最大值宽、光学强度强等特性。通过哺乳动物双杂交测定、活细胞成像、单链探针、生物发光胶囊和生物发光抗体等多种生物检测方法，检验了荧光素酶作为光学读出的实际适用性。我们还通过生物信息学分析进一步确定了 ALuc 的物理性质，并最终讨论了 ALuc 独特性质的详细问题。本研究展示了如何为生物检测创建具有优异光学性质的人工酶，并鼓励研究人员制造具有设计特性和功能的独特人工酶。
