用于低ATP细胞环境中体内成像的红色荧光嵌合萤火虫荧光素酶。
Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments.
作者信息
Branchini Bruce R, Southworth Tara L, Fontaine Danielle M, Kohrt Dawn, Welcome Franceine S, Florentine Catherine M, Henricks Emma R, DeBartolo Demetria B, Michelini Elisa, Cevenini Luca, Roda Aldo, Grossel Martha J
机构信息
Department of Chemistry, Connecticut College, New London, CT 06320, USA.
Department of Chemistry, Connecticut College, New London, CT 06320, USA.
出版信息
Anal Biochem. 2017 Oct 1;534:36-39. doi: 10.1016/j.ab.2017.07.001. Epub 2017 Jul 4.
Abstract
Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O, and added luciferin. Previous Photinus pyralis red-emitting variants with high K values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a K value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the K value in low ATP environments.
摘要
甲虫荧光素酶已被应用于活细胞成像，其中生物发光取决于细胞内ATP、氧气和添加的荧光素的可用性。先前的萤火虫（Photinus pyralis）红色发光变体对ATP具有较高的K值，尽管其相对比活性比诸如叩头虫红色荧光素酶（CBR）等酶高得多，但在活细胞中的表现却令人失望。我们设计了一种荧光素酶变体PLR3，其对ATP的K值与CBR相似，比活性高约2.6倍。在活的HEK293T和HeLa细胞中，红色发光的PLR3比CBR亮约2.5倍，这一改进与K值在低ATP环境中的重要性一致。
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