Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools
Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools
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DOI：
10.1021/acscombsci.7b00081
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发表时间：
2017-09-01
影响因子：
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通讯作者：
Suzuki, Koji
中科院分区：
文献类型：
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作者：
Kim, Sung Bae;Nishihara, Ryo;Suzuki, Koji
具有独特光学性质的人工半胱氨酸酶的制备对生物检测和分子成像具有重要影响。在这项研究中，我们开发了一个新的谱系的ALucs独特的底物偏好提取共识氨基酸的25桡足类萤光素酶序列的天然萤光素酶池的比对。首先使用序列标志生成器创建主序列，从而产生总共11个兄弟序列。系统发育分析表明，新制造的ALucs形成了一个独立的分支，从天然酶中遗传分离，并从我们实验室使用较小的基组产生的先前一系列ALucs中分离。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。新的ALucs的C-末端序列的单残基水平的比较表明，在C-末端的一些氨基酸的光学强度有很大的影响，但在颜色的变化有限。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 fabrication of artificial luciferases (ALucs) with unique optical properties has a fundamental impact on bioassays and molecular imaging. In this study, we developed a new lineage of ALucs with unique substrate preferences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools. The primary sequence was first created with a sequence logo generator resulting in a total of 11 sibling sequences. 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 new lineage of ALucs were strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. A single-residue-level comparison of the C-terminal sequences of new ALucs reveals that some amino acids in the C-terminal ends are greatly influential on the optical intensities but limited in the color variance. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.