Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management.

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.