A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis
Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:95 |
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| Enthalten in: |
Analytical chemistry - 95(2023), 38 vom: 26. Sept., Seite 14341-14349 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Chang, Mu-Shen [VerfasserIn] |
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| Links: |
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| Themen: |
EC 1.11.1.- |
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| Anmerkungen: |
Date Completed 27.09.2023 Date Revised 27.09.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.analchem.3c02684 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM362127964 |
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| 245 | 1 | 2 | |a A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis |
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| 520 | |a Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay | ||
| 650 | 4 | |a Journal Article | |
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| 650 | 7 | |a Horseradish Peroxidase |2 NLM | |
| 650 | 7 | |a EC 1.11.1.- |2 NLM | |
| 650 | 7 | |a Luciferases, Firefly |2 NLM | |
| 650 | 7 | |a EC 1.13.12.7 |2 NLM | |
| 700 | 1 | |a Lee, Chia-Yi |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, En-Shuo |e verfasserin |4 aut | |
| 700 | 1 | |a Chao, Hsuan |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Hsin-Yu |e verfasserin |4 aut | |
| 700 | 1 | |a Chang, Yu-Yen |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Yen-Ling |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Yu-Tung |e verfasserin |4 aut | |
| 700 | 1 | |a Su, Yu-Cheng |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Yeng-Tseng |e verfasserin |4 aut | |
| 700 | 1 | |a Cheng, Tian-Lu |e verfasserin |4 aut | |
| 700 | 1 | |a Yen, Chia-Hung |e verfasserin |4 aut | |
| 700 | 1 | |a Lin, Cheng-Wei |e verfasserin |4 aut | |
| 700 | 1 | |a Huang, Hsin-Kai |e verfasserin |4 aut | |
| 700 | 1 | |a Lin, Wen-Wei |e verfasserin |4 aut | |
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