Recombinant Human Pendrin (SLC26A4) (H723R) is produced using an E.coli-based cell-free expression system, which appears to deliver efficient synthesis and high-quality yields. This full-length protein spans amino acids 1-780 and carries an H723R mutation. It comes with an N-terminal 6xHis-tag to simplify purification. The product shows purity levels greater than 85% based on SDS-PAGE analysis and uses a detergent platform that seems well-suited for maintaining the integral 12 transmembrane domains.
The SLC26A4 gene encodes pendrin, an anion exchanger that handles the transport of iodide and chloride ions across cell membranes. This protein likely plays a crucial role in ion balance within various tissues—particularly the thyroid and inner ear—where it influences processes like hearing and hormone synthesis. Its function and regulation have become significant areas of interest for researchers studying metabolic and auditory pathways.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Based on the provided information, the recombinant Human Pendrin (SLC26A4) with the H723R mutation is expressed using an in vitro E. coli expression system (cell-free system), which generally promotes better protein folding by reducing cellular stress and aggregation. However, Pendrin is a complex membrane protein with 12 transmembrane domains, and the H723R mutation is associated with Pendred syndrome, often leading to misfolding and loss of function in vivo. The protein is full-length (1-780aa) with an N-terminal 6xHis tag, and purity is >85% by SDS-PAGE. Since activity is unverified, the protein cannot be assumed to be correctly folded or bioactive. While the cell-free system may improve folding probability, the inherent challenges of membrane protein folding and the known pathogenic mutation significantly increase the risk of misfolding. Experimental validation (e.g., via circular dichroism for secondary structure, size-exclusion chromatography for oligomeric state, or functional assays for anion transport activity) is essential to confirm folding and bioactivity.
1. Membrane Protein Biochemical Characterization Studies
This recombinant Pendrin (H723R) can be used for biochemical characterization, including studies on protein folding, stability, and detergent interactions using techniques like dynamic light scattering or circular dichroism. The His-tag facilitates purification and immobilization. However, if the protein is misfolded due to the mutation or expression system, the results may not reflect native behavior. Comparative studies with wild-type Pendrin should be interpreted cautiously, as differences may arise from folding artifacts rather than mutation-specific effects.
2. Antibody Development and Epitope Mapping
This application is well-supported. The recombinant protein can serve as an immunogen for generating antibodies, as antibodies may recognize linear epitopes even if the protein is misfolded. The His-tag simplifies screening and validation in ELISA or Western blot. However, if misfolded, antibodies may not recognize conformational epitopes of native Pendrin. Epitope mapping to distinguish between wild-type and H723R forms should include validation against the endogenous protein to ensure specificity.
3. Protein-Protein Interaction Studies Using Pull-Down Assays
The His-tag enables pull-down assays to identify binding partners, but this application is highly dependent on correct folding. If Pendrin is misfolded, interactions may be non-physiological, leading to false positives or negatives. The detergent-solubilized format may not fully replicate the membrane environment, and the H723R mutation could alter binding affinity. This approach should only be pursued after confirming protein folding and activity.
4. Comparative Mutational Analysis Platform
This H723R variant is suitable for comparative studies with wild-type Pendrin to analyze mutation effects on biophysical properties (e.g., expression, solubility). The consistent expression system allows reproducible comparisons. However, if folding is not validated, differences observed may be due to misfolding rather than the mutation itself. It should emphasize that folding must be confirmed to attribute changes to the mutation accurately.
Final Recommendation & Action Plan
Given the uncertainty in folding and bioactivity due to the H723R mutation and membrane protein complexity, it is critical to first validate the protein's conformation using biophysical methods (e.g., size-exclusion chromatography for monodispersity, circular dichroism for secondary structure) and, if possible, functional assays (e.g., anion transport tests). Once folding is confirmed, the protein can be reliably used for comparative mutational analysis and biochemical characterization; if misfolded, focus on applications like antibody development or as a control for folding studies. Avoid protein-protein interaction studies until native conformation is verified. Always include controls such as wild-type Pendrin and use orthogonal methods to validate key findings. Prioritize applications that are less dependent on native folding, such as antibody generation, while proceeding cautiously with biochemical assays.
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