Improved assay sensitivity of an engineered secreted Renilla luciferase
Improved assay sensitivity of an engineered secreted Renilla luciferase
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DOI：
10.1016/s0378-1119(99)00314-5
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发表时间：
1999-09-03
期刊：
影响因子：
3.5
通讯作者：
Escher, A
中科院分区：
文献类型：
--
作者：
Liu, JX;Escher, A
我们以前曾报道构建了一种功能性的Renilla荧光素酶，当融合到人IL-2的信号肽时，哺乳动物细胞可以分泌这种酶。在雷尼拉荧光素酶的氨基酸序列中存在三个预测的半胱氨酸残基，这表明其分泌物中可能含有氧化的巯基，这可能会削弱酶的活性。在这项工作中，构建了四个分泌型Renilla荧光素酶突变体来研究这种可能性：三个荧光素酶突变体，其中不同的半胱氨酸残基被丙氨酸残基取代，以及一个荧光素酶突变体，其中三个半胱氨酸残基都被丙氨酸残基取代。将编码这些突变的荧光素酶的基因以及原始的基因构建体导入猴细胞，并检测细胞培养上清液中的生物发光活性。只有在前蛋白第152位含有半胱氨酸到丙氨酸取代的突变荧光素酶的培养基比含有原始分泌的雷尼拉荧光素酶的培养基有显著的生物发光活性增加。这种光发射的增加部分是由于突变酶的稳定性增强。这种新的酶代表了分泌型Renilla荧光素酶检测用于监测基因表达的灵敏度的显著提高。(C)1999 Elsevier Science B.V.保留所有权利。
We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression. (C) 1999 Elsevier Science B.V. All rights reserved.