Bioluminescent and structural features of native folded Gaussia luciferase
Bioluminescent and structural features of native folded Gaussia luciferase
复制标题
DOI：
10.1016/j.jphotobiol.2018.04.050
复制
发表时间：
2018-06-01
影响因子：
5.4
通讯作者：
Vysotski, Eugene S.
中科院分区：
文献类型：
--
作者：
Larionova, Marina D.;Markova, Svetlana V.;Vysotski, Eugene S.
海洋桡足类动物分泌的荧光素酶负责发光，已被广泛用于细胞内事件的无创成像。桡足动物Gaussia princeps分泌的荧光素酶是一种单亚基蛋白，可催化胆肠嗪氧化而发出蓝光。它由携带一个用于分泌的信号肽的n端可变部分和含有十个高度保守的Cys残基的c端催化结构域组成，假设存在多达五个S-S键。尽管高斯荧光素酶在生物医学研究中得到了广泛的应用，但由于在细菌细胞中获得适当折叠的富含cys的蛋白质的普遍问题，对其生化特性的研究仍然不足。本文报道了利用杆状病毒表达系统在昆虫细胞中产生的适当折叠的高斯荧光素酶的性质。这种高纯度荧光素酶在15-20℃时显示出最高的活性，但在37℃时仅保持类似20%的活性，这可能会妨碍其在体内试验中的应用。GpLuc的生物发光活性在1.0 ~ 1.5 M NaCl浓度范围内达到最大值，且高NaCl浓度增强了荧光素酶对热变性的稳定性，即高斯荧光素酶表现出嗜盐酶的特征。不同药浓度下的荧光素酶生物发光动力学研究表明，高斯荧光素酶与药浓度下的荧光素酶具有良好的协同性（希尔系数-1.8 +/- 0.2;K-0.5-2.14 +/- 0.17 μ M）。我们认为，这种效应是由于所谓的动力学协同作用，由响应底物结合的构象变化而不是两个催化位点的存在所决定的。
The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five S-S bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 degrees C but retains only similar to 20% activity at 37 degrees C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient -1.8 +/- 0.2; K-0.5-2.14 +/- 0.17 mu M). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.