Yasunaga Mayu, Murotomi Kazutoshi, Abe Hiroko, Yamazaki Tomomi, Nishii Shigeaki, Ohbayashi Tetsuya, Oshimura Mitsuo, Noguchi Takako, Niwa Kazuki, Ohmiya Yoshihiro, Nakajima Yoshihiro
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan.
Tsuruga Institute of Biotechnology, TOYOBO Corporation Ltd., Tsuruga, Fukui 914-0047, Japan.
J Biotechnol. 2015 Jan 20;194:115-23. doi: 10.1016/j.jbiotec.2014.12.004. Epub 2014 Dec 18.
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 (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°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-κB after transient treatment with tumor necrosis factor α 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.
使用荧光素酶的报告基因检测广泛应用于监测体外和体内与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性变化的响应，常使用去稳定化序列来降低细胞中荧光素酶蛋白的稳定性，这导致荧光素酶报告基因检测的灵敏度增加。在本研究中，我们鉴定了一个由人钙蛋白酶3（CAPN3）的42个氨基酸残基组成的有效去稳定化序列（称为C9片段）。在37°C进行测量时，来自巴西叩甲Pyrearinus termitilluminans的Emerald Luc（ELuc）通过融合PEST（半衰期从9.8小时降至2.8小时），其半衰期缩短，而C9融合的ELuc的半衰期明显短于PEST融合的ELuc（半衰期=1.0小时）。此外，与人性化的PEST序列相比，萤火虫荧光素酶（luc2）也被C9片段显著去稳定化。这些结果表明，来自CAPN3的C9片段是比PEST序列更有效的去稳定化序列。此外，在用肿瘤坏死因子α短暂处理后，对核因子κB激活动力学的实时生物发光记录显示，C9融合的ELuc的响应明显大于PEST融合的ELuc，这表明使用C9片段实现了一种荧光素酶报告基因检测，其响应速度比PEST序列提供的检测更快。
