First Full-Length Cloning of Human NaV1.7 in S. Cerevisiae-Based Novel D-Crypt^TM Platform for its High-Scale Production
DOI:
https://doi.org/10.48048/tis.2024.7232Keywords:
NaV1.7 Voltage-gated sodium channel, Fluorescence resonance energy transfer, Saccharomyces cerevisiae, Sodium channel blockersAbstract
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-cryptÔ 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-cryptÔ platform. Cloning of NaV1.7 in the D-cryptÔ 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.
HIGHLIGHTS
- 7 is a voltage-gated sodium channel and induces chronic pain
- 7 inhibitors can advance the treatment of chronic pain associated with various diseases
- To characterize interaction of Nav1.7 with its inhibitors, higher yield of the NaV1.7 is required
- D-crypt is a platform designed for the high-yield production of ‘difficult-to-express’ proteins (DTE-Ps) up to 500 L
- Successful cloning of NaV1.7 in D-crypt platform suggest that the D-crypt platform is a suitable platform for expressing full-length functionally active hNaV1.7 channel at larger scale. It will further aid in the screening of NaV1.7 inhibitors
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