In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions
New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, an...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/6/3409 |
| _version_ | 1797240667848572928 |
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| author | Rokhsareh Rozbeh Karl Forchhammer |
| author_facet | Rokhsareh Rozbeh Karl Forchhammer |
| author_sort | Rokhsareh Rozbeh |
| collection | DOAJ |
| description | New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we explored the in vitro protein interaction dynamics of the PII signalling protein using NanoBiT, revealing significant modulation of luminescence signals generated by the interaction between PII and its receptor protein NAGK by 2-oxoglutarate levels. In the current work, we investigated this technology in vivo, to find out whether recombinantly expressed NanoBiT constructs using the NanoLuc large fragment fused to PII and PII-interaction partners NAGK or PipX-fused to the NanoLuc Small BiT are capable of detecting the metabolic fluctuations in <i>Escherichia coli</i>. Therefore, we devised an assay capable of capturing the metabolic responses of <i>E. coli</i> cells, demonstrating real-time metabolic perturbation upon nitrogen upshift or depletion treatments. In particular, the PII-NAGK NanoBitT sensor pair reported these changes in a highly sensitive manner. |
| first_indexed | 2024-04-24T18:11:05Z |
| format | Article |
| id | doaj.art-2d27869c8388474cba9e3f3a57c733b2 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-04-24T18:11:05Z |
| publishDate | 2024-03-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2d27869c8388474cba9e3f3a57c733b22024-03-27T13:46:00ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-03-01256340910.3390/ijms25063409In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein InteractionsRokhsareh Rozbeh0Karl Forchhammer1Interfaculty Institute of Microbiology and Infection Biology, University Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, GermanyInterfaculty Institute of Microbiology and Infection Biology, University Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, GermanyNew protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we explored the in vitro protein interaction dynamics of the PII signalling protein using NanoBiT, revealing significant modulation of luminescence signals generated by the interaction between PII and its receptor protein NAGK by 2-oxoglutarate levels. In the current work, we investigated this technology in vivo, to find out whether recombinantly expressed NanoBiT constructs using the NanoLuc large fragment fused to PII and PII-interaction partners NAGK or PipX-fused to the NanoLuc Small BiT are capable of detecting the metabolic fluctuations in <i>Escherichia coli</i>. Therefore, we devised an assay capable of capturing the metabolic responses of <i>E. coli</i> cells, demonstrating real-time metabolic perturbation upon nitrogen upshift or depletion treatments. In particular, the PII-NAGK NanoBitT sensor pair reported these changes in a highly sensitive manner.https://www.mdpi.com/1422-0067/25/6/3409protein-fragment complementation assayNanoLucluciferase2-oxoglutaratePII-interacting protein X (PipX)N-Acetylglutamate kinase (NAGK) |
| spellingShingle | Rokhsareh Rozbeh Karl Forchhammer In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions International Journal of Molecular Sciences protein-fragment complementation assay NanoLuc luciferase 2-oxoglutarate PII-interacting protein X (PipX) N-Acetylglutamate kinase (NAGK) |
| title | In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions |
| title_full | In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions |
| title_fullStr | In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions |
| title_full_unstemmed | In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions |
| title_short | In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions |
| title_sort | in vivo detection of metabolic fluctuations in real time using the nanobit technology based on pii signalling protein interactions |
| topic | protein-fragment complementation assay NanoLuc luciferase 2-oxoglutarate PII-interacting protein X (PipX) N-Acetylglutamate kinase (NAGK) |
| url | https://www.mdpi.com/1422-0067/25/6/3409 |
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