TNFRSF10B Human, Sf9

What is TNFRSF10B and what is its functional role?

TNFRSF10B (also known as Death Receptor 5, DR5, CD262, KILLER, TRAIL-R2) is a member of the TNF-receptor superfamily of transmembrane proteins. It contains a cytoplasmic "death domain" capable of activating the cell's apoptotic machinery. This receptor is activated by binding to either membrane-anchored or soluble TRAIL/Apo2L, initiating a signaling cascade that leads to programmed cell death. TNFRSF10B is a critical mediator in the extrinsic apoptosis pathway, making it an important target in cancer research due to its potential to selectively induce apoptosis in tumor cells .

What is the significance of Sf9 expression for TNFRSF10B research?

Sf9 refers to Spodoptera frugiperda (fall armyworm) insect cells used as an expression system for recombinant proteins. For TNFRSF10B research, the Sf9 baculovirus expression system offers several advantages: it provides proper eukaryotic post-translational modifications (particularly glycosylation), yields high expression levels, and produces proteins with structural integrity similar to mammalian-expressed proteins. The TNFRSF10B Human, Sf9 preparation specifically refers to human TNFRSF10B produced in this insect cell expression system, which is ideal for functional studies where properly folded and glycosylated protein is required .

What are the structural characteristics of TNFRSF10B Human, Sf9 protein?

TNFRSF10B Human, Sf9 is a single, glycosylated polypeptide chain containing 394 amino acids (positions 56-210 of the native sequence) with a molecular mass of 43.9kDa. On SDS-PAGE analysis, it typically appears at approximately 40-57kDa due to glycosylation. In the recombinant preparation, it is expressed with a 239 amino acid hIgG-His tag at the C-Terminus to facilitate purification. The preparation is purified by proprietary chromatographic techniques to greater than 90% purity as determined by SDS-PAGE .

What are the optimal storage conditions for TNFRSF10B Human, Sf9?

For short-term use (2-4 weeks), TNFRSF10B protein solution can be stored at 4°C. For longer storage periods, it should be kept frozen at -20°C. The standard formulation contains Phosphate Buffered Saline (pH 7.4) and 10% glycerol. For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) to enhance stability. Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity. Aliquoting the protein before freezing is advised for research requiring multiple experimental timepoints .

How can researchers measure the functional activity of TNFRSF10B Human, Sf9?

The functional activity of TNFRSF10B Human, Sf9 can be measured by its ability to inhibit cytotoxicity in assays using Jurkat human T lymphocyte cells. The ED50 (effective dose for 50% inhibition) for this effect is typically ≤5ng/ml with TRAIL. This cytotoxicity inhibition assay serves as the standard functional assessment for TNFRSF10B activity. Alternative methods include binding assays with labeled TRAIL, apoptosis assays in susceptible cell lines, and co-immunoprecipitation studies to assess interaction with downstream signaling components .

What experimental controls should be included when working with TNFRSF10B Human, Sf9?

When designing experiments with TNFRSF10B Human, Sf9, researchers should include:

• Positive controls: Cells known to express functional TRAIL receptors (e.g., Jurkat cells)

• Negative controls: Cell lines with low/no TNFRSF10B expression or TNFRSF10B-knockout cells

• Dose-response curves: Serial dilutions of TNFRSF10B to establish proper working concentrations

• Specificity controls: Other TNF receptor family members to confirm specificity of observed effects

• Vehicle controls: Buffer-only treatments to account for any effects from the storage solution

These controls help ensure experimental validity and assist in troubleshooting unexpected results when working with this protein .

How do TNFRSF10B polymorphisms influence cancer outcomes?

Table 1: TNFRSF10B Polymorphisms Associated with NSCLC Survival

How does TNFRSF10B expression relate to treatment response in cancer patients?

TNFRSF10B/DR5 expression is inducible by cancer therapeutic agents, including platinum agents and taxanes commonly used as adjuvant chemotherapy for lung cancer patients. Upregulation of TNFRSF10B/DR5 can induce programmed cell death or augmentation of TRAIL-induced apoptosis. Research has shown that TNFRSF10B polymorphisms had treatment-specific effects: risk-allele haplotypes exhibited a statistically significant increased risk of death among patients who underwent surgery only, but no significant effects among patients who received surgery and adjuvant chemotherapy. This suggests that TNFRSF10B polymorphisms might serve as predictive biomarkers for determining which patients would benefit from adjuvant chemotherapy following surgical resection .

What methodological approaches are used to study TNFRSF10B-mediated apoptosis in tumor models?

Several methodologies are employed to study TNFRSF10B-mediated apoptosis in tumor models:

• Apoptosis assays: Annexin V/PI staining followed by flow cytometry to quantify early and late apoptotic cells

• Caspase activity measurements: Fluorometric or colorimetric assays for caspase-8, caspase-10, and caspase-3 activation

• Death receptor expression analysis: Immunoblotting, immunohistochemistry, and flow cytometry to assess TNFRSF10B levels

• Genetic approaches: siRNA knockdown, CRISPR/Cas9 gene editing, or overexpression systems to modulate TNFRSF10B levels

• Combination studies: Testing TNFRSF10B agonists with chemotherapeutic agents to identify synergistic interactions

These approaches help elucidate the mechanisms of TNFRSF10B-mediated apoptosis and identify potential therapeutic strategies targeting this pathway .

How can TNFRSF10B Human, Sf9 be utilized in drug discovery programs?

TNFRSF10B Human, Sf9 protein can be employed in various drug discovery applications:

• High-throughput screening: Development of binding assays to identify small molecules that modulate TNFRSF10B activity

• Structure-based drug design: Using purified protein for crystallography studies to determine three-dimensional structure

• Antibody development: Generation of therapeutic antibodies targeting TNFRSF10B

• TRAIL mimetics testing: Evaluation of synthetic TRAIL variants or peptide mimetics for receptor activation

• Combination therapy optimization: Identifying compounds that sensitize cancer cells to TRAIL-induced apoptosis

The recombinant protein serves as a valuable tool for these applications due to its high purity and consistent activity profile .

What is the functional significance of TNFRSF10B genetic variants in cancer biology?

TNFRSF10B genetic variants can affect protein function through several mechanisms, although the precise functional impacts are still being elucidated. In silico analyses have revealed that the rs1047275 locus is located in binding sites for miR-379 and miR-473, potentially regulating TNFRSF10B expression. These microRNAs have been shown to play roles in drug response in lung cancer and colon cancer metastasis. If TNFRSF10B SNPs functionally impact protein expression or activity, this could result in deficient apoptotic responses in tumor cells, providing a tumor survival advantage and ultimately adversely impacting patient survival. Allelic losses in the chromosomal region (8p21–23) harboring TNFRSF10B have been reported as frequent events in several cancers including lung cancer, suggesting that attenuated TNFRSF10B/DR5-induced apoptosis, possibly due to genetic variations, is associated with cancer development and progression .

What technical challenges exist when working with TNFRSF10B in experimental systems?

Researchers face several technical challenges when working with TNFRSF10B:

• Protein stability: Death receptors like TNFRSF10B can be unstable and lose activity during storage or handling

• Oligomerization requirements: Effective activation often requires receptor clustering, which can be difficult to replicate in vitro

• Cell type specificity: Response to TNFRSF10B activation varies greatly between cell types

• Background apoptosis: Distinguishing receptor-specific from non-specific cell death in experimental systems

• Reproducibility issues: Variations in glycosylation patterns between protein batches can affect functional studies

Addressing these challenges requires careful experimental design, appropriate controls, and standardized protocols to ensure reproducible and meaningful results .

How is TNFRSF10B being explored as a biomarker for personalized cancer treatment?

TNFRSF10B is emerging as a potential germline biomarker for personalized cancer treatment strategies. Studies have demonstrated that specific TNFRSF10B polymorphisms and haplotypes correlate with survival outcomes in NSCLC patients, particularly in relation to treatment response. The discovery that risk haplotypes exhibited increased mortality in surgical-only patients but not in patients receiving adjuvant chemotherapy suggests that TNFRSF10B genetic testing could help identify which early-stage patients would benefit from adjuvant chemotherapy. This represents a shift toward using germline biomarkers, rather than just tumor markers, for treatment stratification. Ongoing research is exploring the predictive value of TNFRSF10B variants across different cancer types, treatment regimens, and patient populations to develop more personalized therapeutic approaches .

What approaches can be used to study TNFRSF10B signaling complex formation?

Advanced techniques for studying TNFRSF10B signaling complex formation include:

• Proximity ligation assays: Detecting protein-protein interactions in situ with high sensitivity

• FRET/BRET techniques: Measuring real-time interactions between TNFRSF10B and adapter proteins

• Mass spectrometry-based proteomics: Identifying components of the death-inducing signaling complex (DISC)

• Super-resolution microscopy: Visualizing receptor clustering and complex formation at the nanoscale level

• Hydrogen-deuterium exchange mass spectrometry: Mapping protein interaction interfaces

These methods help elucidate the dynamics and composition of the signaling complexes formed upon TNFRSF10B activation, which is crucial for understanding receptor functionality in normal and disease states .