Expression of Nav1.7 - Premas Biotech

FEATURED CASE STUDY

Expression of Nav1.7

Share this article

Background:

Voltage-gated sodium channels are transmembrane proteins responsible for propagating the electrical signal in neurons, cardiac, skeletal muscle, and neuroendocrine cells. Sodium channel Nav1.7 is preferentially expressed in peripheral neurons. Nav1.7 is a validated target for pain treatment in humans. The principal subunit of this channel is a protein of >200 kDa, the alpha subunit, which consists of four large domains of internal homology. The subunit forms the pore and functions in voltage-dependent activation and inactivation, sensitivity to toxins, and the modulation by cAMP kinase and protein kinase. These discoveries sparked hope for millions suffering from chronic pain: a new and promising drug target had emerged.

Problem/Rationale:

The protein contains oligomeric conformation, a high odd number of cysteine and proline residues, hydrophobic, and therefore was difficult to express. Previous studies have shown the expression of Nav1.7 in mammalian vectors but were not scalable and cost-effective.

Premas has developed a robust process to express the full-length membrane-anchored alpha subunit of Nav1.7 in the D-cryptᵀᴹ platform.

Solution/Approach:

This protein was produced in an in vitro yeast expression system, which preserves posttranslational modification and correct conformational folding necessary for its biological function. Briefly, the gene was cloned into a proprietary episomal expression vector: pYRE100. Then, the construct was transformed into protease deficient proprietary S. cerevisiae host strain PYPD. Expression scouting was performed using various parameters, and the best clone was selected for expression analysis (Figure 1) and localization studies(Figure 2). The functionality of this protein has also been measured or validated using well-known Nav1.7 inhibitors.

Measurable Impact

1) Expression of the functionally active and properly folded alpha subunit of Nav1.7 protein.
2) D-cryptᵀᴹ platform of Premas can supply Nav1.7 at a larger scale for preclinical and clinical studies.
3) The developed protein could be used to screen Nav1.7 antagonists, which have a future in treating most pain syndromes.
4) We have further opportunities of doing codon optimization in terms of strain engineering for the enhanced expression in S. cerevisiae and scale-ups.

Figure 1: Confocal microscopy showing surface expression of NaV1.7

Expression of Highly Active Protein in E. coli for Diagnostics Applicatio

The production of recombinant enzymes has led to the development of many diagnostics strategies in various medical fields. The increasing demand for recombinant proteins has created the need to develop efficient protein expression techniques to increase the productivity of the product.

Expression of Full-Length Neuraminidase (NA)

Below is the case study describing the expression of full-length neuraminidase in the protease-deficient strain of Baker’s yeast, S. cerevisiae of D-Crypt™. The platform provides an ideal eukaryotic environment for expressing recombinant proteins retaining their correct structure, function, and post-translational modifications resembling those of humans. We used an array of proprietary expression vectors with designed upstream sequences for enhanced expression of neuraminidase.

Purification Optimization and Large-Scale Production of a Recombinant Protein fr

The recombinant protein is a shortened form of the native isolate, requiring multiple tests and regulatory submission characterization. Some bacteria produce toxins that show specific activity against certain insect species. This specificity is mainly due to a unique binding site in the insects' midgut epithelium membrane, where the protein inserts and causes damage to the wall.