Borisova Vasilisa V, Frank Ludmila A, Markova Svetlana V, Burakova Ludmila P, Vysotski Eugene S
Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia.
Photochem Photobiol Sci. 2008 Sep;7(9):1025-31. doi: 10.1039/b807271j. Epub 2008 Jun 17.
The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity. The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.
来自海洋桡足类长腹水蚤的重组腔肠素依赖性荧光素酶（同工型MLuc164和MLuc39）在大肠杆菌细胞中以包涵体形式表达，溶解于6 M盐酸胍中，并在为含有分子内二硫键的蛋白质开发的条件下进行折叠。其中一种（MLuc39）以高产率获得了活性单体形式。发现荧光素酶生物发光不仅由游离腔肠素引发，而且在添加Ca2+时由穆氏海肾的Ca2+依赖性腔肠素结合蛋白（CBP）引发。使用CBP作为“底物”可提供更高的发光强度，同时降低背景水平。高纯度的MLuc39在低至阿托摩尔时即可检测到，线性范围超过5个数量级。MLuc39对加热和化学修饰也具有很高的稳定性；荧光素酶的化学合成生物素化衍生物保留了35-40%的初始活性。在结合Ca2+调节的光蛋白奥贝林和长腹水蚤荧光素酶的模型串联生物发光固相微分析中，证明了荧光素酶作为体外生物发光报告分子的适用性。
