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.

中文翻译：

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从天然荧光素酶库中制备新的人工荧光素酶谱系。

具有独特光学性质的人工荧光素酶（ALucs）的制造对生物测定和分子成像具有根本性的影响。在这项研究中，我们通过从天然萤光素酶库中可用的25种co足类萤光素酶序列的比对中提取共有氨基酸，开发了具有独特底物偏好的ALucs新谱系。首先使用序列徽标生成器创建主要序列，从而产生总共11个同级序列。系统发育分析表明，新构建的ALucs形成了一个独立的分支，与天然萤光素酶以及我们实验室使用较小基数集生产的先前系列ALucs遗传分离。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定的底物选择性。新ALucs C末端序列的单残基水平比较显示，C末端的某些氨基酸对光强度有很大影响，但颜色变化受到限制。这种方法的成功指导了如何为生物发光成像和分析工程化和功能化海洋荧光素酶。