Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia.
Biochem Biophys Res Commun. 2012 Jan 6;417(1):98-103. doi: 10.1016/j.bbrc.2011.11.063. Epub 2011 Nov 22.
The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.
实时成像技术在活细胞中对于理解细胞内事件至关重要。为此，生物发光报告基因已被引入作为敏感和方便的工具。来自桡足类长刺水蚤的虫荧光素酶（MLuc）是一种依赖腔肠素的荧光素酶，含有用于分泌的天然信号肽。我们报道了具有氨基酸序列 N 端可变部分缺失的高活性 MLuc 突变体。MLuc 变体在大肠杆菌细胞中产生，转化为活性蛋白，并进行了表征。我们证明，与野生型酶相比，截断的 MLuc 具有显著增加的生物发光活性，但基本保留了其他特性。MLuc 的一种截断变体在 HEK 293 细胞中瞬时表达。结果清楚地表明，截断的虫荧光素酶非常适合作为分泌型报告基因，与野生型酶相比，可提高检测灵敏度。
