Chang Mu-Shen, Lee Chia-Yi, Chang Yu-Yen, Li Pin-Jie, Wu Hsin-Yu, Liu En-Shuo, Huang Hsin-Kai, Lin Wen-Wei
College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
J Biol Eng. 2025 Apr 22;19(1):37. doi: 10.1186/s13036-025-00508-w.
A light-producing secretory protein that is collectible through the supernatant of a culture medium is essential in a cell-based reporter gene system and allows for real-time monitoring of upstream events of a promoter. Compared to other secretory luciferases, Cypridina luciferase (CLuc) coupled with vargulin emits the brightest signal; however, the signal half-life suffers constantly from the fast oxidation process of the substrate, resulting in a rapid signal depletion, which makes the detection signal short and unstable. In this study, we aimed to develop a new reporter gene system with a more stable signal and lower cost, whilst retaining sensitivity comparable to the CLuc reporter gene system. To this end, we genetically engineered horseradish peroxidase (HRP) to be secreted with mammalian cells. The secreted form HRP (sHRP) was then used as a proof-of-concept of real-time cell signaling management. First, we made sure that HRP retained its enzymatic function with our genetic engineering process and confirmed that it was collectable and suitable for side-by-side comparison with CLuc. sHRP showed comparable sensitivity, 7 to 80 times more signal half-life compared to CLuc, and precisely reported NF-κB-regulated promoter in response to stimulation with TNF-α. sHRP was not affected by multiple cell culturing media and was calculated to be at least 9 times cheaper than the CLuc reporter gene system. Thus, sHRP offers new insight into the reporter gene system for drug screening and intracellular signaling management and provides a precise, sustainable and affordable operating environment.
一种可通过培养基上清液收集的发光分泌蛋白在基于细胞的报告基因系统中至关重要，它能够实时监测启动子的上游事件。与其他分泌型荧光素酶相比，结合了腔肠素的海萤荧光素酶（CLuc）发出的信号最亮；然而，由于底物的快速氧化过程，信号半衰期持续缩短，导致信号迅速耗尽，使得检测信号短暂且不稳定。在本研究中，我们旨在开发一种新的报告基因系统，其信号更稳定且成本更低，同时保持与CLuc报告基因系统相当的灵敏度。为此，我们通过基因工程使辣根过氧化物酶（HRP）在哺乳动物细胞中分泌。然后，将分泌形式的HRP（sHRP）用作实时细胞信号管理的概念验证。首先，我们确保HRP在我们的基因工程过程中保留其酶功能，并确认它可收集且适合与CLuc进行并行比较。sHRP显示出相当的灵敏度，与CLuc相比，信号半衰期长7至80倍，并能精确报告在肿瘤坏死因子-α刺激下NF-κB调控的启动子。sHRP不受多种细胞培养基的影响，经计算其成本至少比CLuc报告基因系统便宜9倍。因此，sHRP为药物筛选和细胞内信号管理的报告基因系统提供了新的见解，并提供了一个精确、可持续且经济实惠的操作环境。
