| Autor: |
Arora, Kajal, Roy, Sourav Singha, Kumar, Sudhir, Rastogi, Ruchir, Prattipati, Mahesh, Gupta, Reeshu, Mehrotra, Nupur, Kundu, Prabuddha |
| Předmět: |
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| Zdroj: |
Trends in Sciences; Feb2024, Vol. 21 Issue 2, p1-8, 8p |
| Abstrakt: |
NaV1.7, a voltage-gated sodium channel, induces chronic pain. Current research on the discovery of inhibitors against NaV1.7 is advancing with the hope of treating chronic pain conditions in patients suffering from various diseases. However, to characterize NaV1.7 and inhibitor interactions, a higher yield of expression is required to obtain sufficient protein. Molecular cloning of hNaV1.7 was done using the homologous recombination method in our novel S. cerevisiae-based D-crypt™ platform. The platform was designed for the high-yield production of 'difficult-to-express' proteins (DTE-Ps) up to 500 L. The changes in the cell membrane potential of hNaV1.7 were determined using a fluorescence resonance energy transfer (FRET) assay with tetracaine (hNaV1.7 inhibitor). Expression analysis of hNaV1.7 showed 2 bands of size 250 and 280 kDa that were absent in untransfected yeast cells. Immunofluorescence images revealed the presence of hNaV1.7 on the membrane of hNaV1.7-expressing yeast cells. Tetracaine exhibited concentration-dependent inhibition of the FRET ratio, with the order of potency (IC50 = 0.46 µM) being approximately the same as previously reported, suggesting the functionality of the channel protein expressed in the D-crypt™ platform. Cloning of NaV1.7 in the D-crypt™ platform will help to scale up the production of channel proteins, which will ultimately help in the structural and functional characterization of the binding interactions between toxins and the NaV1.7 channel to identify more specific NaV1.7 inhibitors. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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