In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions.
Int J Mol Sci
; 25(6)2024 Mar 17.
Article
en En
| MEDLINE
| ID: mdl-38542383
ABSTRACT
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 Escherichia coli. Therefore, we devised an assay capable of capturing the metabolic responses of E. coli 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.
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Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Synechococcus
Idioma:
En
Revista:
Int J Mol Sci
Año:
2024
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Suiza