TY - JOUR
T1 - A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis
AU - Chang, Mu Shen
AU - Lee, Chia Yi
AU - Liu, En Shuo
AU - Chao, Hsuan
AU - Wu, Hsin Yu
AU - Chang, Yu Yen
AU - Liu, Yen Ling
AU - Chen, Yu Tung
AU - Su, Yu Cheng
AU - Wang, Yeng Tseng
AU - Cheng, Tian Lu
AU - Yen, Chia Hung
AU - Lin, Cheng Wei
AU - Huang, Hsin Kai
AU - Lin, Wen Wei
N1 - Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/9/26
Y1 - 2023/9/26
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.3c02684
DO - 10.1021/acs.analchem.3c02684
M3 - Article
AN - SCOPUS:85174341577
SN - 0003-2700
VL - 95
SP - 14341
EP - 14349
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 38
ER -