Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
Abstract 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) co...
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2025-04-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-025-00508-w |
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| author | Mu-Shen Chang Chia-Yi Lee Yu-Yen Chang Pin-Jie Li Hsin-Yu Wu En-Shuo Liu Hsin-Kai Huang Wen-Wei Lin |
| author_facet | Mu-Shen Chang Chia-Yi Lee Yu-Yen Chang Pin-Jie Li Hsin-Yu Wu En-Shuo Liu Hsin-Kai Huang Wen-Wei Lin |
| author_sort | Mu-Shen Chang |
| collection | DOAJ |
| description | Abstract 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. Graphical Abstract |
| format | Article |
| id | doaj-art-a2b761e30b7b41818685dfa6e7d015b3 |
| institution | OA Journals |
| issn | 1754-1611 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biological Engineering |
| spelling | doaj-art-a2b761e30b7b41818685dfa6e7d015b32025-08-20T02:30:24ZengBMCJournal of Biological Engineering1754-16112025-04-0119111410.1186/s13036-025-00508-wGenetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity managementMu-Shen Chang0Chia-Yi Lee1Yu-Yen Chang2Pin-Jie Li3Hsin-Yu Wu4En-Shuo Liu5Hsin-Kai Huang6Wen-Wei Lin7College of Life Science, Kaohsiung Medical UniversityCollege of Life Science, Kaohsiung Medical UniversityGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical UniversityDepartment of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical UniversityGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical UniversityGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical UniversityDepartment of Medical Laboratory, Kaohsiung Armed Forces General HospitalCollege of Life Science, Kaohsiung Medical UniversityAbstract 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. Graphical Abstracthttps://doi.org/10.1186/s13036-025-00508-wSecreted form horseradish peroxidaseReporter gene systemReal-time monitoringStable signalLow cost |
| spellingShingle | Mu-Shen Chang Chia-Yi Lee Yu-Yen Chang Pin-Jie Li Hsin-Yu Wu En-Shuo Liu Hsin-Kai Huang Wen-Wei Lin Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management Journal of Biological Engineering Secreted form horseradish peroxidase Reporter gene system Real-time monitoring Stable signal Low cost |
| title | Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management |
| title_full | Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management |
| title_fullStr | Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management |
| title_full_unstemmed | Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management |
| title_short | Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management |
| title_sort | genetically engineered secretory horseradish peroxidase is a sensitive stable and affordable non lytic reporter gene system for real time promoter activity management |
| topic | Secreted form horseradish peroxidase Reporter gene system Real-time monitoring Stable signal Low cost |
| url | https://doi.org/10.1186/s13036-025-00508-w |
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