TNFR2 Human, Sf9

What is the structural composition of human TNFR2 and how does it differ from TNFR1?

Human TNFR2 belongs to the TNF receptor family and exhibits a more limited cellular distribution compared to TNFR1. While TNFR1 is expressed on nearly all bodily cells, TNFR2 is predominantly found on specific immune cells (CD4+ and CD8+ lymphocytes), certain CNS cells, endothelial cells, T-regulatory cells, microglia, neuron subtypes, oligodendrocytes, cardiac myocytes, thymocytes, islets of Langerhans, and human mesenchymal stem cells .

Structurally, TNFR2 preferentially binds to transmembrane-bound TNF, and crystal structure analysis has shown that these interactions result in the formation of aggregates on the cell surface that promote signaling . Unlike TNFR1, which typically signals cell death through activation of the adaptor proteins TRADD and FADD, TNFR2 signaling primarily relies on TRAF2 and activation of the pro-survival transcription factor nuclear factor-kB (NFkB) .

What are the advantages of using Sf9 insect cells for human TNFR2 expression?

Sf9 insect cells provide several advantages for expressing human TNFR2, particularly for structural and functional studies. Insect cell expression systems like Sf9 offer post-translational modifications that are closer to mammalian systems than bacterial expression systems. For complex receptors like TNFR2, Sf9 cells can facilitate proper protein folding, disulfide bond formation, and glycosylation patterns that are essential for maintaining the receptor's native conformation and functionality.

The baculovirus expression system in Sf9 cells allows for high-yield production of recombinant TNFR2, which is particularly beneficial for structural studies, binding assays, and agonist/antagonist screening. Additionally, Sf9 cells can be grown in suspension culture, enabling scalable protein production for various experimental applications.

How can I optimize the purification of human TNFR2-ECD from Sf9 expression systems?

For optimal purification of human TNFR2 extracellular domain (ECD) from Sf9 cells, a multi-step purification process is recommended:

• Begin with affinity chromatography using a properly tagged TNFR2-ECD construct (such as His-tagged or Fc-fusion proteins)

• Implement additional purification steps using ion-exchange chromatography to separate proteins based on charge differences

• Finalize with size-exclusion chromatography to achieve high purity and remove aggregates

When designing the TNFR2-ECD construct for Sf9 expression, consider incorporating a cleavable signal peptide to enhance secretion into the culture medium. This approach can simplify the initial purification steps by allowing harvesting from the supernatant rather than cell lysates. Monitor glycosylation patterns of the purified protein, as appropriate glycosylation is crucial for proper folding and function of TNFR2.

What methodologies are most effective for measuring binding affinity between human TNFR2 and TNF-α?

Several sophisticated methodologies can be employed to measure binding affinity between human TNFR2 and TNF-α, with surface plasmon resonance (SPR) being particularly effective:

• Surface Plasmon Resonance (SPR): Using platforms such as Biacore 3000, TNF-α can be immobilized on a sensor chip using amine-coupling methods. TNFR2-ECD is then serially diluted (typically between 1 and 32 μM) in binding buffer and injected over the chip at a controlled flow rate (30 μl/min). The association and dissociation phases are monitored for approximately 60 seconds each, and data are analyzed using evaluation software by fitting to a 1:1 Langmuir binding model .

• Enzyme-Linked Immunosorbent Assay (ELISA): Microtiter plates can be coated with varying concentrations of TNFR2-ECD in PBS overnight, followed by blocking with BSA. TNF-α is then added to each well, and binding is detected using specific antibodies. This approach allows for quantitative assessment of binding under different conditions .

• Förster Resonance Energy Transfer (FRET): For studying interactions in living cells, TNFR2 and its binding partners can be tagged with appropriate fluorophores (such as EYFP-TNFR2 as acceptor and ECFP-tagged binding partners as donors). Acceptor photobleaching FRET microscopy can then measure the relative FRET efficiency to quantify interactions .

Table 1: Comparative Analysis of Binding Affinity Measurement Techniques for TNFR2

How does IL-17RD influence TNFR2 signaling, and what implications does this have for experimental design?

Research has revealed that TNFR2 forms a heteromer with interleukin-17 receptor D (IL-17RD, also known as Sef) to activate NF-κB signaling. This interaction is specific to TNFR2 and not TNFR1, presenting important considerations for experimental design .

The association between TNFR2 and IL-17RD leads to mutual receptor aggregation and TRAF2 recruitment, which subsequently activates the downstream NF-κB signaling cascade. Notably, this interaction is enhanced in the presence of TNF-α, with the IL-17RD·TNFR2 complex increasing upon TNF-α stimulation .

When designing experiments to study TNFR2 signaling in Sf9 systems or other cellular contexts, researchers should consider:

• Co-expressing IL-17RD with TNFR2 to recapitulate physiological signaling conditions

• Implementing co-immunoprecipitation assays to verify complex formation

• Utilizing FRET-based approaches to visualize interactions in real-time

• Including appropriate controls to distinguish TNFR2-specific effects from TNFR1-mediated responses

Immunostaining experiments have shown that endogenous IL-17RD and TNFR2 co-localize on the cell membrane, and this co-localization is enhanced and shifts to intracellular perinuclear compartments after TNF-α treatment . This spatial reorganization is an important experimental consideration when studying TNFR2 trafficking and signaling dynamics.

What are the challenges in distinguishing TNFR2-specific signals from TNFR1 in experimental systems?

Distinguishing TNFR2-specific signals from TNFR1 presents several challenges in experimental systems, particularly because cells that express TNFR2 typically also express TNFR1, with the ratio of expression varying according to cell type and functional role .

Key methodological approaches to overcome these challenges include:

• Receptor-specific agonists: Utilize TNFR2-selective agonistic antibodies that do not activate TNFR1 signaling

• Genetic approaches: Implement CRISPR/Cas9 to generate TNFR1 knockouts while maintaining TNFR2 expression

• Domain-specific constructs: Express truncated or chimeric receptors in Sf9 cells to isolate TNFR2-specific domains

• Downstream signaling analysis: Monitor TRAF2 recruitment and NFκB activation patterns, which differ between the receptors

What optimization strategies improve functional TNFR2 expression in Sf9 cells?

Optimizing functional TNFR2 expression in Sf9 cells requires attention to several parameters:

• Codon optimization: Human-to-insect codon optimization can significantly improve translation efficiency and protein yield

• Signal peptide selection: Testing different signal peptides (both insect and mammalian-derived) can enhance secretion and proper membrane localization

• Expression timing: Harvest timing is critical; TNFR2 quality and yield may peak at different time points post-infection (typically 48-72 hours)

• Culture conditions: Optimize temperature (27-28°C is standard, but lower temperatures may improve folding), pH, and dissolved oxygen levels

• Infection multiplicity: Titrate the multiplicity of infection (MOI) to determine optimal viral load for expression

For membrane-bound TNFR2, consider using specialized Sf9 cell lines engineered for improved mammalian protein expression. For secreted TNFR2-ECD production, implement fed-batch cultures with protein stabilizing additives to maximize yield.

How can researchers effectively analyze TNFR2 oligomerization states from Sf9-expressed proteins?

TNFR2 forms oligomeric structures upon ligand binding, a critical step in its signaling mechanism. To effectively analyze these oligomerization states from Sf9-expressed proteins, researchers can employ multiple complementary techniques:

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This technique separates protein complexes based on size while simultaneously determining absolute molecular weight, providing insights into the stoichiometry of TNFR2 complexes

• Native PAGE: Non-denaturing gel electrophoresis allows visualization of intact TNFR2 complexes

• Chemical Crosslinking: Before SDS-PAGE analysis, chemical crosslinkers can "freeze" protein interactions, preserving oligomeric states

• Analytical Ultracentrifugation: Sedimentation velocity and equilibrium experiments provide detailed information about size, shape, and association constants

When expressed in Sf9 cells, TNFR2 has been shown to preferentially bind transmembrane-bound TNF, and crystal structure analysis has demonstrated that these interactions result in the formation of aggregates on the cell surface that promote signaling . Capturing and characterizing these oligomeric states is essential for understanding TNFR2 function.

What are the critical quality control parameters for validating TNFR2 expressed in Sf9 cells?

Validating the quality of TNFR2 expressed in Sf9 cells requires comprehensive assessment of several parameters:

• Structural integrity: Circular dichroism spectroscopy can confirm proper secondary structure composition

• Glycosylation profile: Mass spectrometry analysis of glycan patterns, which may differ from mammalian cells but must support proper folding

• Thermal stability: Differential scanning fluorimetry to assess protein stability and the effects of different buffer conditions

• Functional binding: Surface plasmon resonance to verify TNF-α binding with expected kinetics and affinity

• Oligomerization capacity: Size exclusion chromatography to confirm appropriate oligomeric states form upon ligand binding

Table 2: Quality Control Checklist for Sf9-Expressed Human TNFR2

How can Sf9-expressed TNFR2 be utilized for screening potential therapeutic agonists?

Sf9-expressed human TNFR2 provides an excellent platform for screening potential therapeutic agonists, particularly for autoimmune disease applications. Research has shown that TNFR2 agonism has been associated with selective death of autoreactive T cells in type 1 diabetes and with induction of T-regulatory cells .

A comprehensive screening approach includes:

• Primary binding assays: Implement high-throughput binding assays using labeled Sf9-expressed TNFR2-ECD

• Functional reporter systems: Develop Sf9 cells co-expressing TNFR2 with relevant human signaling components and luminescent/fluorescent reporters

• Structural screening: Utilize crystallography with Sf9-expressed TNFR2 to determine binding sites of potential agonists

• Signaling validation: Confirm candidates promote appropriate TRAF2 recruitment and NFκB activation

When designing the screening system, it's critical to distinguish between TNFR2-selective agonists and those that might cross-react with TNFR1, as TNFR1 activation is associated with systemic toxicity . The therapeutic potential of TNFR2-selective agonists lies in their ability to target more restricted cell populations compared to TNFR1, which is expressed throughout the body.

What approaches can resolve contradictory data regarding TNFR2 signaling pathways in different cell types?

Research has revealed seemingly contradictory findings regarding TNFR2 signaling across different cell types and disease contexts. To resolve these contradictions, researchers should implement several strategic approaches:

• Context-specific expression systems: Express TNFR2 with cell-type specific co-factors in Sf9 cells

• Signaling component analysis: Systematically analyze the expression and activation status of downstream signaling components

• Temporal signaling resolution: Implement time-course experiments to capture signaling dynamics

• Receptor complex composition: Investigate how different binding partners (like IL-17RD) modify signaling outcomes

Research has shown that TNFR2 forms a heteromer with IL-17RD to activate NF-κB signaling, but this interaction may be context-dependent . Additionally, TNFR2 signaling varies across different physiological conditions – it offers protective roles in several disorders, including autoimmune disease, heart disease, and demyelinating disorders .

When contradictory data emerges, researchers should consider both cell-intrinsic factors (expression levels of signaling components) and cell-extrinsic factors (inflammatory environment, presence of other cytokines) that might influence TNFR2 signaling outcomes.

How can researchers design experiments to investigate the therapeutic potential of TNFR2 agonism versus antagonism in autoimmune diseases?

Designing experiments to investigate the therapeutic potential of TNFR2 agonism versus antagonism requires careful consideration of disease context and cellular targets. The search results indicate that TNFR2 agonism can selectively destroy autoreactive T cells but not healthy T cells in blood samples from type I diabetes patients, as well as in multiple sclerosis, Graves, and Sjogren's disease models .

A comprehensive experimental design should include:

• Comparative agonist/antagonist screening: Use Sf9-expressed TNFR2 to identify and characterize selective modulators

• Cell-type specific responses: Test effects on isolated autoreactive T cells versus regulatory T cells

• Mechanistic validation: Confirm proposed mechanism (NFκB dysregulation making autoreactive T cells selectively vulnerable to TNF-induced apoptosis)

• Disease-specific modeling: Adapt experiments to reflect the specific TNFR2 defects associated with each autoimmune disease

Table 3: Experimental Design Framework for TNFR2 Therapeutic Modulation

What strategies can address low yield or poor stability of TNFR2 expressed in Sf9 cells?

Researchers frequently encounter challenges with low yield or poor stability when expressing human TNFR2 in Sf9 cells. These issues can be addressed through several strategic approaches:

• Construct optimization:

  • Add stabilizing mutations identified through computational analysis

  • Include fusion partners (such as thioredoxin or SUMO) to enhance solubility

  • Design truncated constructs that retain functional domains while removing unstable regions

• Expression conditions optimization:

  • Reduce expression temperature to 19-22°C during protein production phase

  • Supplement media with chemical chaperones like glycerol or arginine

  • Implement a biphasic production process (growth at 27°C, expression at lower temperature)

• Purification workflow modifications:

  • Add stabilizing ligands during purification

  • Include protease inhibitors throughout all purification steps

  • Optimize buffer composition with stabilizing additives (glycerol, specific ions)

When working with membrane-bound TNFR2, consider expressing in Sf9 cells as a GPI-anchored construct, which can improve surface expression while allowing for release using phospholipase C for easier purification.

How can researchers reconcile differences between TNFR2 activity in Sf9-expressed systems versus mammalian contexts?

Differences in TNFR2 activity between Sf9-expressed systems and mammalian contexts arise from several factors that must be systematically addressed:

• Post-translational modification differences:

  • Characterize glycosylation patterns using mass spectrometry

  • Assess differences in disulfide bond formation

  • Evaluate the impact of these differences on receptor function

• Co-factor requirements:

  • Identify missing mammalian co-factors through complementation experiments

  • Co-express key human signaling components in Sf9 cells

  • Test the addition of mammalian membrane extracts to Sf9-expressed TNFR2

• Signaling pathway reconstitution:

  • Implement step-wise reconstruction of signaling components

  • Monitor critical interactions, such as TNFR2 association with IL-17RD, which has been shown to enhance TNFR2 signaling

Research has shown that TNFR2 relies on TRAF2 and activation of the pro-survival transcription factor nuclear factor-kB (NFkB) . When designing experiments, researchers should consider whether these downstream signaling components are adequately represented in their insect cell systems.

What analytical techniques can resolve conflicting structural data about TNFR2 ligand-binding domains?

Resolving conflicting structural data about TNFR2 ligand-binding domains requires integration of multiple complementary analytical techniques:

• High-resolution structural analysis:

  • X-ray crystallography of TNFR2-ECD expressed in Sf9 cells with and without bound ligands

  • Cryo-electron microscopy to visualize different conformational states

  • NMR spectroscopy for dynamic binding interface mapping

• Mutagenesis studies:

  • Alanine scanning mutagenesis of predicted binding interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify protected regions upon binding

  • Cross-linking coupled with mass spectrometry to identify interaction points

• Computational approaches:

  • Molecular dynamics simulations to compare different proposed binding modes

  • Ensemble modeling to reconcile seemingly conflicting structural data

  • Free energy calculations to quantify binding contributions of specific residues

Research has shown that TNFR2 preferentially binds transmembrane-bound TNF, and crystal structure analysis has demonstrated that these interactions result in the formation of aggregates on the cell surface that promote signaling . When analyzing conflicting structural data, researchers should consider whether differences might reflect distinct functional states or oligomerization states of the receptor.