Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Methods Mol Biol. 2021;2274:43-51. doi: 10.1007/978-1-0716-1258-3_5.
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.
本方案介绍了一种新型的人工荧光素酶（ALucs），具有独特的光学特性，可用于哺乳动物细胞成像。最初的候选序列是使用序列标志生成器创建的，通过从公共数据库中天然荧光素酶池中的 25 个桡足类荧光素序列的比对中提取共识氨基酸，总共得到了 11 个同源序列。系统发育分析表明，新合成的 ALucs 形成了一个独立的分支，在遗传上与天然荧光素酶以及我们实验室使用较小基序集产生的先前一系列 ALucs 分离。该方案还举例说明了新型 ALucs 在具有特定底物选择性的活哺乳动物细胞中具有很强的发光性，对天然腔肠素具有特异性。这种方法的成功为海洋荧光素酶的生物发光成像和分析提供了设计和功能化的指导。
