A New Lineage of Artificial Luciferases for Mammalian Cell Imaging
A New Lineage of Artificial Luciferases for Mammalian Cell Imaging
复制标题
用于哺乳动物细胞成像的新型人工荧光素酶
DOI：
10.1007/978-1-0716-1258-3_5
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Fujii Rika
中科院分区：
文献类型：
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作者：
Kim Sung-Bae;Fujii Rika
本协议介绍了一个新的谱系的人工核糖核酸酶（ALucs）具有独特的光学性质的哺乳动物细胞成像。首先用序列标识生成器创建主要候选序列，通过从公共数据库中天然荧光素酶池中可用的25个桡足类荧光素酶序列的比对中提取共有氨基酸，得到总共11个同胞序列。系统发育分析表明，新制造的ALucs形成了一个独立的分支，从天然酶和我们实验室使用较小的基组产生的先前一系列ALucs中遗传分离。该方案还证实了ALucs的新谱系在活的哺乳动物细胞中强烈发光，对天然腔肠素具有特异性底物选择性。这种方法的成功指导了如何工程化和功能化海洋生物发光成像和分析。
The present protocol introduces a new lineage of artificial luciferases (ALucs) with unique optical properties for mammalian cell imaging. The primary candidate sequence was first created with a sequence logo generator, resulting in a total of 11 sibling sequences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools in public databases. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set. The protocol also exemplifies that the new lineage of ALucs was strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.