Recombinant Mouse Tumor necrosis factor receptor superfamily member 10B (Tnfrsf10b), partial (Active)

What is mouse TNFRSF10B and what is its biological role?

Mouse TNFRSF10B (also known as DR5, KILLER, or CD262) is a member of the TNF receptor superfamily containing an intracellular death domain. It functions as a receptor for TNF-related apoptosis-inducing ligand (TRAIL) and plays a crucial role in transducing apoptosis signals in various cell types . The protein contains multiple functional domains including an extracellular TRAIL-binding region, a transmembrane domain, and an intracellular death domain that recruits adaptor proteins like FADD to initiate the apoptotic cascade.

In normal physiological conditions, TNFRSF10B participates in immune surveillance and maintenance of cellular homeostasis. Upon binding to its ligand TRAIL, TNFRSF10B forms a death-inducing signaling complex (DISC) that activates initiator caspases, particularly caspase-8, leading to the activation of downstream executioner caspases and ultimately resulting in programmed cell death. This mechanism is particularly significant in cancer research as TRAIL selectively induces apoptosis in transformed cells while generally sparing normal cells .

Mouse TNFRSF10B exhibits high homology with its human counterpart but possesses specific sequence variations that researchers should consider when designing cross-species experiments or translational studies. The recombinant partial active form typically contains the extracellular domain that is sufficient for TRAIL binding and subsequent biological activity assays.

How does recombinant mouse TNFRSF10B differ from the native protein?

Recombinant mouse TNFRSF10B represents a partial sequence of the native protein, typically encompassing amino acids 53-177, which corresponds to the extracellular domain responsible for ligand binding . This fragment retains the critical functional regions necessary for TRAIL interaction but lacks the transmembrane and intracellular domains present in the full-length endogenous protein. The recombinant version is often produced with expression tags, such as a C-terminal human Fc (hFc) tag, which facilitates purification and detection while potentially enhancing stability .

Several key differences between recombinant and native TNFRSF10B include:

The recombinant protein's biological activity is typically assessed by its ability to inhibit TRAIL-mediated cytotoxicity, with potent activity observed at concentrations less than 1 μg/ml when tested in L-929 mouse fibroblast cells . This inhibitory function stems from its ability to competitively bind TRAIL, preventing the ligand from interacting with membrane-bound receptors.

What are the key methods for verifying the identity and purity of recombinant mouse TNFRSF10B?

SDS-PAGE remains the primary method for assessing purity, with high-quality recombinant mouse TNFRSF10B typically showing greater than 95% purity . For optimal resolution, a 12-15% gel is recommended with both reducing and non-reducing conditions to evaluate potential disulfide-bonded aggregates. Silver staining provides higher sensitivity than Coomassie for detecting minor contaminants.

Western blotting using specific antibodies against mouse TNFRSF10B or the tag (e.g., anti-hFc) confirms the protein's identity. When using antibodies against the native protein, bands should appear at approximately 40.9 kDa for the hFc-tagged version . Mass spectrometry analysis provides definitive identification and can detect post-translational modifications or truncations.

Endotoxin contamination can significantly impact cellular assays, particularly those involving immune cells. Limulus Amebocyte Lysate (LAL) testing should confirm levels below 1.0 EU/μg . Functional verification through bioactivity assays, specifically the inhibition of TRAIL-mediated cytotoxicity in L-929 cells, provides crucial evidence of proper folding and biological relevance .

For proteins intended for structural studies, additional characterization via circular dichroism or differential scanning calorimetry can verify proper secondary structure formation and thermal stability, essential parameters for ensuring consistency between batches and experimental reliability.

What are the optimal storage and reconstitution conditions for recombinant mouse TNFRSF10B?

Proper storage and reconstitution of recombinant mouse TNFRSF10B are critical for maintaining its structural integrity and biological activity across extended research timelines. Recombinant TNFRSF10B is typically supplied as a lyophilized powder, which offers superior stability compared to liquid formulations during shipping and long-term storage . The lyophilized product should be stored at -20°C and protected from light and moisture to prevent degradation.

For reconstitution, it is essential to briefly centrifuge the vial prior to opening to bring all contents to the bottom and minimize product loss . The protein should be reconstituted in sterile, deionized water to achieve a concentration between 0.1-1.0 mg/mL . This initial reconstitution should be performed gently, avoiding vigorous vortexing that can cause protein denaturation and aggregation. Instead, gentle swirling or slow pipetting is recommended until the lyophilized cake is completely dissolved, which may take 5-10 minutes at room temperature.

For long-term storage of the reconstituted protein, the addition of a cryoprotectant is strongly recommended. Adding glycerol to a final concentration of 5-50% significantly enhances stability during freeze-thaw cycles . The reconstituted protein should be aliquoted into small volumes suitable for single experiments to avoid repeated freeze-thaw cycles, which progressively reduce activity. These aliquots should be stored at -20°C for routine use or -80°C for extended storage periods.

Working solutions should be prepared fresh on the day of experimentation in appropriate buffers compatible with the intended assay system. For cellular assays, dilution in serum-free media or phosphate-buffered saline supplemented with 0.1-0.5% bovine serum albumin can enhance protein stability and reduce non-specific binding.

How can recombinant mouse TNFRSF10B be used to study TRAIL-mediated apoptosis pathways?

Recombinant mouse TNFRSF10B serves as a valuable tool for dissecting TRAIL-mediated apoptosis pathways through multiple experimental approaches. As a soluble decoy receptor, it can competitively inhibit TRAIL binding to cell surface receptors, providing a method to selectively block this pathway in complex biological systems.

For pathway interrogation, researchers can employ recombinant TNFRSF10B in dose-response experiments to determine the threshold concentration required for inhibiting TRAIL-induced apoptosis. Typically, concentrations below 1 μg/ml are sufficient to observe significant inhibition in sensitive cell lines like L-929 mouse fibroblasts . This approach allows researchers to quantitatively assess TRAIL dependency in various experimental models and compare sensitivity across different cell types or disease states.

In more complex experimental systems, recombinant TNFRSF10B can be used to distinguish between TRAIL-dependent and TRAIL-independent apoptosis mechanisms. By selectively neutralizing TRAIL activity in models with multiple potential death inducers (such as tumor microenvironment studies or inflammatory disease models), researchers can determine the relative contribution of TRAIL signaling to the observed phenotype.

The protein can also be immobilized on surfaces (such as tissue culture plates or sensor chips) to study binding kinetics and receptor-ligand interactions using techniques like surface plasmon resonance. This approach provides quantitative data on association and dissociation rates, helping to characterize mutations or species-specific variations in binding properties.

For advanced studies of signaling dynamics, combining recombinant TNFRSF10B treatments with time-course analyses of downstream effectors (caspase activation, mitochondrial membrane potential changes, or nuclear fragmentation) enables precise mapping of signaling kinetics and threshold responses in the apoptotic cascade.

What controls should be included when working with recombinant mouse TNFRSF10B in functional assays?

Rigorous experimental design incorporating appropriate controls is essential for generating reliable and interpretable data when working with recombinant mouse TNFRSF10B. A comprehensive control strategy should address protein specificity, functional activity validation, and experimental system variables.

Primary controls should include a vehicle control (buffer only) to establish baseline measurements and rule out buffer-mediated effects. An isotype control protein (e.g., irrelevant protein with the same tag) at equivalent concentrations helps distinguish specific TNFRSF10B-mediated effects from non-specific protein or tag-related artifacts. For inhibition assays, a positive control using a validated TRAIL neutralizing antibody provides a reference standard for comparison.

Concentration-dependent responses should be demonstrated through a dose-response curve spanning at least three orders of magnitude (e.g., 0.01-10 μg/ml). This approach establishes the ED50 value, which should be consistent with reported values of less than 1 μg/ml for inhibition of TRAIL-mediated cytotoxicity in L-929 cells .

For systems employing genetic manipulations (knockdown, overexpression, or CRISPR-mediated editing), include both wild-type cells and appropriate genetic controls (e.g., scrambled shRNA, empty vector) to account for manipulation-specific effects. When possible, validation across multiple cell lines reduces the likelihood of cell line-specific artifacts and strengthens the generalizability of findings.

Time-course experiments should include both early (1-6 hours) and late (24-72 hours) timepoints to capture both rapid signaling events and delayed phenotypic outcomes, as TRAIL-mediated effects may manifest with different kinetics depending on the cellular context and endpoint measured.

How do mouse and human TNFRSF10B variants differ in research applications?

Understanding the differences between mouse and human TNFRSF10B is crucial for designing translational studies and interpreting cross-species experiments. These differences span sequence homology, expression patterns, ligand interactions, and downstream signaling characteristics.

Functionally, both proteins can bind TRAIL, but with different affinities and downstream consequences. Human TNFRSF10B demonstrates activity by reducing TNF production induced by lipopolysaccharide in human peripheral blood mononuclear cells , while mouse TNFRSF10B inhibits TRAIL-mediated cytotoxicity in L-929 mouse fibroblast cells . These distinct activity assays reflect the optimized experimental systems for each species variant.

For research applications, these differences necessitate careful consideration when selecting the appropriate variant:

When designing mechanistic studies, researchers should be aware that mouse models may not fully recapitulate human TRAIL pathway dynamics due to these differences. Studies aimed at therapeutic development should incorporate both species variants at early stages to identify potential species-specific effects that might impact translation from preclinical to clinical applications.

What are the critical considerations when designing TRAIL-mediated cell death experiments using recombinant TNFRSF10B?

Designing robust TRAIL-mediated cell death experiments with recombinant TNFRSF10B requires careful attention to multiple experimental parameters that can significantly influence outcomes and interpretability. These considerations span protein handling, experimental design, cellular context, and data analysis approaches.

Protein concentration and activity verification are paramount. Fresh reconstitution of TNFRSF10B before each critical experiment ensures consistent activity, and pre-incubation of the recombinant protein with TRAIL (at established molar ratios) before addition to cells allows for formation of the inhibitory complex. The functional activity should be verified in a known responsive system (e.g., L-929 cells) as a quality control measure for each new lot or after extended storage .

Cell culture conditions significantly impact TRAIL pathway sensitivity. Serum components can contain factors that alter TRAIL signaling, so serum concentration should be standardized and lot-tested. Cell density affects both TRAIL sensitivity and TNFRSF10B inhibitory efficiency, with optimal results typically achieved at 60-80% confluence. The timing of treatment relative to plating is also critical, with most protocols recommending treatment 24 hours after plating to allow for recovery from trypsinization stress.

For co-treatment or sequential treatment protocols, careful timing is essential:

For mechanistic studies, combining TNFRSF10B inhibition with genetic approaches (siRNA knockdown of pathway components) or small molecule inhibitors (caspase inhibitors, necrostatin-1) helps delineate the specific contribution of distinct death pathways and identify potential compensatory mechanisms.

How can researchers troubleshoot inconsistent results with TNFRSF10B in cellular assays?

Inconsistent results when working with recombinant TNFRSF10B can stem from multiple sources related to protein quality, experimental conditions, cellular variables, and technical execution. A systematic troubleshooting approach addressing each potential factor can help resolve variability and enhance experimental reproducibility.

Protein-related factors are common sources of variation. Repeated freeze-thaw cycles progressively reduce activity, with significant deterioration typically observed after 3-5 cycles. Preparing single-use aliquots with glycerol (5-50% final concentration) minimizes this issue . Protein aggregation, often undetectable by visual inspection, can be assessed via dynamic light scattering or size-exclusion chromatography. Fresh reconstitution in appropriate buffers (typically 20 mM PB, 150 mM NaCl, pH 7.4) maintains optimal protein conformation .

Cell culture variables frequently contribute to inconsistency. Mycoplasma contamination alters cellular response to death signals and should be regularly tested. Cell passage number affects TRAIL pathway component expression, with late-passage cells often showing altered sensitivity; maintaining cells within a defined passage window (typically passages 5-20) improves consistency. Cell cycle synchronization before treatment may be necessary, as TRAIL sensitivity varies across cell cycle phases.

Technical execution requires standardization across experiments:

Cellular heterogeneity, particularly in cancer cell lines, can drive variability. Consider single-cell analyses or cell sorting to isolate specific populations based on TNFRSF10B expression levels. For adherent cells, standardize harvesting procedures, as trypsinization can temporarily alter surface receptor levels and signaling.

When inconsistency persists despite addressing these factors, biological variables may be responsible. TRAIL sensitivity fluctuates with metabolic state, so standardizing culture nutrients and seeding density helps normalize cellular metabolism. Environmental stressors (hypoxia, oxidative stress) profoundly affect TRAIL pathway components, necessitating strict control of incubation conditions. Finally, epigenetic drift in cultured cell lines can gradually alter phenotypes; periodically returning to early-passage frozen stocks helps maintain consistent cellular responses.

How is TNFRSF10B involved in cancer pathways and what are its key research applications in oncology?

TNFRSF10B holds significant importance in cancer biology as a receptor capable of selectively inducing apoptosis in transformed cells while generally sparing normal cells, making it a promising target for cancer research. The protein's involvement in cancer pathways is multifaceted, spanning tumor suppression, immune surveillance, and therapeutic resistance mechanisms.

The TNFRSF10B pathway intersects with major oncogenic signaling networks. P53, a critical tumor suppressor, directly regulates TNFRSF10B expression, with DNA damage inducing TNFRSF10B upregulation in p53-competent cells . This connection provides a mechanistic link between genotoxic stress and apoptotic response. Additionally, NF-κB signaling, often dysregulated in cancer, modulates TNFRSF10B sensitivity through regulation of anti-apoptotic proteins.

Key research applications of recombinant TNFRSF10B in cancer research include:

• Pathway Analysis: Using recombinant TNFRSF10B to inhibit TRAIL-mediated apoptosis allows researchers to quantify the contribution of this pathway to cell death induced by various anti-cancer agents, helping identify synergistic combinations.

• Biomarker Development: Correlating tumor TNFRSF10B expression levels with treatment response helps identify patient subgroups likely to benefit from TRAIL-targeting therapies.

• Resistance Mechanism Identification: Comparing TRAIL sensitivity before and after development of therapeutic resistance, with TNFRSF10B as a tool to probe pathway integrity, reveals specific resistance mechanisms.

• Immunotherapy Research: Investigating how TNFRSF10B signaling intersects with immune checkpoint pathways and contributes to immune surveillance provides insights for designing more effective immunotherapeutic approaches.

For translational applications, research has focused on developing TRAIL receptor agonists that selectively trigger cancer cell death through TNFRSF10B. Recombinant TNFRSF10B serves as a valuable control in these studies, helping distinguish on-target from off-target effects of these experimental therapeutics.

What experimental systems best model TNFRSF10B function in tumor microenvironments?

Modeling TNFRSF10B function in tumor microenvironments requires experimental systems that capture the complexity of cancer tissues beyond simple cell line models. These advanced systems enable researchers to study how TNFRSF10B signaling operates within the context of heterogeneous cell populations, three-dimensional architecture, and dynamic cell-cell interactions characteristic of actual tumors.

Three-dimensional organoid cultures derived from primary tumor tissues represent one of the most physiologically relevant in vitro models. These self-organizing structures maintain critical features of the original tumor, including cellular heterogeneity, spatial organization, and cell-cell contacts that influence TNFRSF10B signaling. When treated with recombinant TNFRSF10B in combination with TRAIL, these organoids allow for assessment of differential sensitivity across distinct cellular subpopulations within the same tumor.

Co-culture systems incorporating multiple cell types provide insights into how tumor-stroma interactions modulate TNFRSF10B function:

Microfluidic devices and organ-on-chip platforms offer advanced capabilities for studying TNFRSF10B in dynamic microenvironments. These systems enable precise control over spatial arrangement of multiple cell types, introduction of mechanical forces (e.g., fluid shear stress), and creation of chemical gradients that more accurately mimic in vivo conditions. When coupled with live-cell imaging, these platforms allow real-time visualization of TRAIL-induced apoptosis and the modulatory effects of recombinant TNFRSF10B.

For in vivo modeling, genetically engineered mouse models with manipulated TNFRSF10B expression provide systemic insights into pathway function. Patient-derived xenograft (PDX) models maintain the heterogeneity and genetic features of the original human tumor and can be used to test how recombinant TNFRSF10B modulates response to various therapeutic agents under physiologically relevant conditions.

Single-cell analysis technologies (scRNA-seq, mass cytometry) applied to these model systems enable the identification of distinct cellular subpopulations with differential TNFRSF10B expression or pathway activity, providing unprecedented resolution of heterogeneous responses within complex tissues.

How can recombinant TNFRSF10B be used to screen potential cancer therapeutics?

Recombinant TNFRSF10B offers versatile applications in screening potential cancer therapeutics, serving both as a tool for pathway interrogation and as a model for therapeutic development. These screening approaches span from high-throughput initial screens to detailed mechanistic investigations of promising candidates.

In pathway-focused screening, recombinant TNFRSF10B can identify compounds that enhance or restore TRAIL sensitivity in resistant cancer cells. By comparing cell viability in the presence of TRAIL alone versus TRAIL plus candidate compounds, with and without recombinant TNFRSF10B as a pathway inhibitor, researchers can determine whether compounds act specifically through the TRAIL-TNFRSF10B axis or through alternative mechanisms. This discriminatory approach helps prioritize compounds with well-defined mechanisms of action.

For combination therapy development, a matrix-based screening approach incorporating recombinant TNFRSF10B provides valuable insights:

This systematic approach enables quantification of how candidate compounds influence TRAIL pathway activity, with recombinant TNFRSF10B serving as a critical control to confirm pathway specificity. Compounds that show synergy with TRAIL that is reversible by recombinant TNFRSF10B can be prioritized as TRAIL pathway sensitizers.

For more mechanistic investigations, recombinant TNFRSF10B can be employed in conjunction with protein-protein interaction assays (co-immunoprecipitation, proximity ligation) to determine whether candidate compounds alter the interaction between TRAIL and its receptors or between death receptors and downstream adaptor proteins. Surface plasmon resonance or biolayer interferometry incorporating immobilized recombinant TNFRSF10B can quantitatively assess how compounds affect binding kinetics and affinities.

In advanced screening platforms, recombinant TNFRSF10B can be incorporated into microfluidic devices or organoid systems to evaluate compounds under more physiologically relevant conditions. By comparing drug responses in the presence and absence of recombinant TNFRSF10B across multiple patient-derived samples, researchers can identify biomarkers predicting which patient populations might benefit most from TRAIL pathway-targeting therapeutics.

What are the optimal antibodies and methods for detecting TNFRSF10B in different experimental systems?

Detecting TNFRSF10B across various experimental systems requires selecting appropriate antibodies and methodologies tailored to the specific application and sample type. The choice of detection strategy significantly impacts sensitivity, specificity, and the ability to distinguish between different protein forms or localization patterns.

For immunoblotting applications, monoclonal antibodies targeting the extracellular domain of TNFRSF10B typically provide higher specificity than polyclonal alternatives. When examining mouse TNFRSF10B in cell lysates, antibodies should be validated to detect the expected molecular weight bands of approximately 44-52 kDa under reducing conditions . For recombinant mouse TNFRSF10B with an hFc tag, a band at approximately 40.9 kDa should be observed . PVDF membranes generally yield better results than nitrocellulose for TNFRSF10B detection, with optimal antibody concentrations typically ranging from 0.5-2 μg/mL .

Flow cytometry enables quantitative assessment of cell surface TNFRSF10B expression across heterogeneous populations:

Immunohistochemistry requires careful optimization for detecting TNFRSF10B in tissue sections. Antigen retrieval methods significantly impact staining quality, with citrate buffer (pH 6.0) generally yielding better results than EDTA-based solutions. For mouse tissue sections, blocking endogenous mouse IgG with specialized blocking reagents prevents non-specific binding when using mouse-derived primary antibodies.

For detecting recombinant TNFRSF10B in solution or binding assays, ELISA-based approaches offer high sensitivity. Sandwich ELISA using a capture antibody against the hFc tag and a detection antibody against the TNFRSF10B domain enables specific quantification of the recombinant protein. This approach typically achieves detection limits in the low ng/mL range when optimized.

Advanced imaging approaches such as proximity ligation assay (PLA) or fluorescence resonance energy transfer (FRET) can detect TNFRSF10B interactions with binding partners like TRAIL or downstream adaptors like FADD. These methods require careful selection of antibody pairs with compatible species origin and epitope targeting to minimize steric hindrance that could interfere with detection.

How can researchers distinguish between TNFRSF10B isoforms and post-translational modifications?

Distinguishing between TNFRSF10B isoforms and identifying post-translational modifications presents significant technical challenges that require specialized approaches beyond standard detection methods. These distinctions are crucial for understanding the protein's functional heterogeneity in different cellular contexts and disease states.

TNFRSF10B exists in multiple isoforms resulting from alternative splicing, with transcript variants encoding functionally distinct proteins. Western blotting can partially distinguish these isoforms based on molecular weight differences, with full-length TNFRSF10B appearing at approximately 44-52 kDa . Using gradient gels (4-20%) improves resolution of closely sized variants. Isoform-specific antibodies, when available, provide the most definitive discrimination, though these require careful validation against recombinant standards of each isoform.

Reverse transcription PCR with isoform-specific primers offers a complementary approach at the mRNA level. Designing primers spanning unique exon junctions enables specific amplification of distinct transcript variants. Quantitative RT-PCR or digital PCR provides accurate quantification of the relative abundance of each isoform across experimental conditions.

Post-translational modifications (PTMs) significantly impact TNFRSF10B function and detection. Common PTMs include:

Mass spectrometry-based approaches provide the most comprehensive characterization of TNFRSF10B proteoforms. After immunoprecipitation to enrich for TNFRSF10B, techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) can identify specific PTM sites and isoform-specific peptides in a single analysis. Top-down proteomics, analyzing intact protein rather than peptide fragments, offers additional insights into combinatorial modifications that may co-occur on individual protein molecules.

For spatial analysis of different TNFRSF10B variants, super-resolution microscopy combined with proximity ligation assays can visualize the subcellular distribution of specific isoforms or modified forms. This approach is particularly valuable for understanding how modification patterns influence receptor clustering and signaling complex formation at the single-molecule level.

What are the most effective methods for quantifying TNFRSF10B-ligand interactions and downstream signaling?

Quantifying TNFRSF10B-ligand interactions and downstream signaling requires sophisticated methodologies that span from biophysical binding assays to functional signaling readouts. These complementary approaches provide a comprehensive understanding of both the initial receptor-ligand engagement and the resulting signal transduction events.

Surface plasmon resonance (SPR) and biolayer interferometry (BLI) represent gold standard methods for quantifying binding kinetics between recombinant TNFRSF10B and its ligands. These label-free techniques enable real-time measurement of association and dissociation rates (kon and koff), as well as the equilibrium dissociation constant (KD). For optimal results, recombinant mouse TNFRSF10B should be immobilized on sensor surfaces via the hFc tag using anti-human IgG capture, preserving the native orientation of the TRAIL-binding domain.

Fluorescence-based interaction assays offer alternatives suitable for high-throughput screening:

For cellular systems, quantifying downstream signaling provides functional readouts of TNFRSF10B activation. Proximal signaling events can be assessed through co-immunoprecipitation of DISC components (FADD, caspase-8) following TRAIL stimulation, with and without recombinant TNFRSF10B competition. Western blotting for cleaved caspase-8 (p43/41 and p18 fragments) provides a direct measure of initiator caspase activation, while cleaved caspase-3 and PARP cleavage indicate execution of the apoptotic program.

Time-resolved analysis using phospho-specific antibodies against key signaling nodes (e.g., JNK, p38 MAPK) via western blotting or bead-based multiplex assays captures the kinetics of non-apoptotic signaling downstream of TNFRSF10B. These approaches benefit from standardization using recombinant proteins as positive controls.

For high-content screening applications, automated microscopy with multiplexed fluorescent reporters enables simultaneous assessment of multiple pathway components at the single-cell level. This approach is particularly valuable for heterogeneous samples where population averages may obscure important subpopulation responses.

Functional genomics approaches using CRISPR screens or RNAi libraries can identify critical nodes in TNFRSF10B signaling by correlating genetic perturbations with altered responses to TRAIL. These unbiased approaches often reveal unexpected pathway components that may represent novel therapeutic targets or biomarkers.