Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3
Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3
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DOI：
10.1016/j.jbiotec.2014.12.004
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发表时间：
2015-01-20
影响因子：
4.1
通讯作者：
Nakajima, Yoshihiro
中科院分区：
文献类型：
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作者：
Yasunaga, Mayu;Murotomi, Kazutoshi;Nakajima, Yoshihiro
使用荧光素酶的记者分析方法被广泛用于监测与体内和体外基因表达相关的细胞事件。为了提高荧光素酶报告基因对基因表达急剧变化的反应，经常使用不稳定的序列来降低荧光素酶蛋白在细胞中的稳定性，这导致了荧光素酶报告分析的灵敏度增加。在这项研究中，我们发现了一个由42个氨基酸残基组成的人类钙蛋白3(CAPN3)的有效失稳序列(称为C9片段)。在37℃的温度下，融合害虫(t(1/2)=9.8~2.8h)可缩短白背飞虱翡翠荧光素酶(ELuc)的半衰期(t(1/2)=9.8~2.8h)，而融合C9的ELuc半衰期(t(1/2)=1.0h)显著短于融合害虫的ELuc(t(1/2)=1.0h)。此外，与人源化的害虫序列相比，C9片段也显著破坏了萤火虫荧光素酶(Luc2)的稳定性。这些结果表明，CAPN3的C9片段是一个比PEST序列更强的失稳序列。此外，实时生物发光记录核因子-kappaB在肿瘤坏死因子α瞬时处理后的激活动力学表明，C9融合ELuc的反应显著高于PEST融合ELuc，表明使用C9片段实现了一种比PEST序列提供更快反应速度的荧光素酶报告基因检测。(C)2014爱思唯尔B.V.保留所有权利。
Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t(1/2) = 9.8 to 2.8 h), the half-life of C9-fused ELuc was significantly shorter (t(1/2) = 1.0 h) than that of PEST-fused ELuc when measurements were conducted at 37 degrees C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-kappa B after transient treatment with tumor necrosis factor alpha revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence. (C) 2014 Elsevier B.V. All rights reserved.