Kim Sung Bae, Nishihara Ryo, Citterio Daniel, Suzuki Koji
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST) , 16-1 Onogawa, Tsukuba 305-8569, Japan.
Department of Applied Chemistry, Faculty of Science and Technology, Keio University , 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
ACS Comb Sci. 2017 Sep 11;19(9):594-599. doi: 10.1021/acscombsci.7b00081. Epub 2017 Aug 9.
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.
具有独特光学性质的人工荧光素酶（ALucs）的制备对生物测定和分子成像具有根本性的影响。在本研究中，我们通过从天然荧光素酶库中可用的25种桡足类荧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新型ALucs谱系。首先使用序列标志生成器创建一级序列，共产生11个姊妹序列。系统发育分析表明，新制备的ALucs形成一个独立的分支，在遗传上与天然荧光素酶以及我们实验室先前使用较小基础集产生的一系列ALucs分离。新型ALucs谱系在活的哺乳动物细胞中具有强烈的发光能力，对天然腔肠素具有特定的底物选择性。对新型ALucs C端序列的单残基水平比较表明，C末端的一些氨基酸对光强度有很大影响，但对颜色变化的影响有限。这种方法的成功为如何改造海洋荧光素酶并使其功能化以用于生物发光成像和测定提供了指导。
