Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China.
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China; Department of Laboratory Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing, China; Laboratory for Diagnosis and Treatment of Infectious Diseases Integrated Traditional Chinese and Western Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing, China.
J Biotechnol. 2022 Sep 20;357:100-107. doi: 10.1016/j.jbiotec.2022.08.005. Epub 2022 Aug 11.
Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.
半胱天冬氨酸蛋白酶（caspases）是一类进化上保守的半胱氨酸蛋白酶家族，在程序性细胞死亡和炎症中发挥关键作用。在检测 caspase 活性的方法中，基于荧光素酶的生物传感器在基因编码和检测方便性方面具有优势。在本研究中，我们构建了基于 NanoLuc 荧光素酶的新型 caspase 生物传感器。这种传感器，命名为 NanoLock，采用暗到亮的工作模式，借助于从 HiBiT 突变而来的 NanoLuc 淬灭肽（HiBiT-R/D）。优化后的 NanoLock 对蛋白酶具有高信噪比（S/N）的响应，在 HEK293 细胞中对烟草蚀纹病毒蛋白酶的激活倍数为 1233 倍，体外对 caspase 3 的诱导倍数超过 500 倍。我们构建了用于检测 caspase 1、3、6、7、8、9 和 10 的 NanoLocks，在 HEK293 细胞中的检测表明，这些传感器在 S/N 和方便性方面优于商业试剂盒。我们进一步建立了稳定表达 NanoLock-casp 6 的细胞系，并为该细胞系在 caspase 6 调节剂的高通量筛选中的应用提供了概念验证。
