Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate
The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:40 |
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| Enthalten in: |
Sheng wu gong cheng xue bao = Chinese journal of biotechnology - 40(2024), 2 vom: 25. Feb., Seite 496-506 |
| Sprache: |
Chinesisch |
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| Beteiligte Personen: |
Zhang, Rui [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 20.02.2024 Date Revised 20.02.2024 published: Print Citation Status MEDLINE |
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| doi: |
10.13345/j.cjb.230502 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM368598934 |
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| 245 | 1 | 0 | |a Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate |
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro | ||
| 650 | 4 | |a English Abstract | |
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a SARS-CoV-2 | |
| 650 | 4 | |a baicalein | |
| 650 | 4 | |a dimerization-dependent red fluorescent protein | |
| 650 | 4 | |a ensitrelvir | |
| 650 | 4 | |a fluorogenic substrate | |
| 650 | 4 | |a main protease | |
| 650 | 7 | |a Red Fluorescent Protein |2 NLM | |
| 650 | 7 | |a 3C-like proteinase, SARS-CoV-2 |2 NLM | |
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| 650 | 7 | |a Fluorescent Dyes |2 NLM | |
| 650 | 7 | |a Peptides |2 NLM | |
| 650 | 7 | |a Protease Inhibitors |2 NLM | |
| 650 | 7 | |a Coronavirus 3C Proteases |2 NLM | |
| 650 | 7 | |a EC 3.4.22.28 |2 NLM | |
| 700 | 1 | |a Yan, Haohao |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Zhicheng |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Xiaoli |e verfasserin |4 aut | |
| 700 | 1 | |a Yan, Gangan |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Xiaoping |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Yunyu |e verfasserin |4 aut | |
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