TNFR Human, Sf9

What are TNF receptors and their significance in research?

TNF receptors consist of two main types: TNFR1 (p55 or TNFRSF1A) and TNFR2 (p75 or TNFRSF1B). These receptors bind tumor necrosis factor-alpha (TNF-α), a major inflammatory cytokine critically involved in immune system homeostasis. The TNF/TNFR system plays dual roles in health and disease - mediating beneficial effects in inflammation and host defense while potentially contributing to pathological conditions like sepsis, cachexia, and autoimmune diseases .

TNFRs are involved in multiple cellular processes including cell proliferation, survival, differentiation, apoptosis, and immune organ development. TNFR1 signaling has been extensively studied and responds to broad inflammatory stimulation in various diseases through NF-κB activation . In contrast, TNFR2 functions more selectively in specific conditions such as certain kidney diseases, requiring co-factors for NF-κB activation . Understanding these receptors provides opportunities for targeted therapeutic interventions in chronic inflammatory and autoimmune conditions.

Why use Sf9 cells for human TNFR expression?

Sf9 insect cells offer several methodological advantages for expressing human TNFRs:

• Post-translational modifications: Sf9 cells perform many essential post-translational modifications required for functional mammalian proteins, particularly important for receptor ectodomains.

• High expression yields: The baculovirus expression system in Sf9 cells typically produces substantial amounts of recombinant proteins with proper folding, critical for structural and functional studies.

• Secretion capability: With appropriate signal sequences (such as gp67), Sf9 cells efficiently secrete proteins into the culture medium, simplifying downstream purification.

• Scalability: Sf9 cultures can be scaled up for larger protein production needs while maintaining consistent quality.

The established methodology for TNFR expression in Sf9 cells involves:

• Constructing expression vectors containing the human TNFR extracellular domain (ECD) with a modified N-terminal gp67 secretion signal sequence and C-terminal His6 tag

• Transforming these constructs into bacterial DH10Bac cells

• Transfecting the resulting bacmid into Sf9 cells with Cellfectin II Reagent

• Harvesting and amplifying the low-titer viruses

• Infecting large-scale Sf9 cultures (typically at 2×10^6 cells/ml)

• Collecting the protein-containing supernatant 48-72 hours post-infection

How do TNFR1 and TNFR2 differ functionally in research contexts?

Understanding the distinct properties of TNFR1 and TNFR2 is essential for experimental design and data interpretation:

In renal tubular epithelial cells (RTECs), TNFR2 is expressed at significantly higher levels than TNFR1, making these cells particularly valuable for studying TNFR2-specific signaling . This differential expression pattern highlights the importance of cell type selection when designing TNFR experiments.

How should researchers optimize TNFR expression in Sf9 cells?

Optimizing human TNFR expression in Sf9 cells requires attention to several methodological parameters:

Vector design considerations:

• For human TNFR1, use residues 22-211 of the extracellular domain

• For human TNFR2, use residues 23-255 of the extracellular domain

• Include a modified N-terminal gp67 secretion signal sequence

• Add a C-terminal His6 tag for purification purposes

Infection parameters:

• Use high-titer virus (typically third passage) for protein production

• Optimal cell density for infection is 2×10^6 cells/ml

• Harvest supernatant containing secreted protein 48-72 hours post-infection

Expression verification:

• Perform small-scale test expressions before scaling up

• Monitor protein expression via Western blot analysis of supernatant samples

• Analyze protein quality via gel filtration chromatography to assess oligomeric state

Following these guidelines enables production of properly folded, functional human TNFR extracellular domains suitable for downstream applications including binding studies, structural analysis, and development of receptor-selective ligands.

What purification strategies yield high-quality TNFR proteins from Sf9 cultures?

A multi-step purification approach is recommended for obtaining high-quality human TNFR proteins from Sf9 cell culture supernatants:

• Initial processing:

  • Harvest supernatant 48-72 hours post-infection

  • Concentrate and buffer-exchange to HEPES-buffered saline (10 mM HEPES, pH 7.2, 150 mM NaCl)

• Immobilized metal affinity chromatography (IMAC):

  • Capture His-tagged protein using nickel resin

  • Wash with buffer containing 20 mM imidazole to remove non-specifically bound proteins

  • Elute with buffer containing 500 mM imidazole

• Size exclusion chromatography:

  • Further purify protein using Superdex 200 column

  • Run in HBS buffer (10 mM HEPES, pH 7.2, 150 mM NaCl)

  • Collect and pool fractions containing properly folded TNFR

This protocol has successfully yielded purified TNFR ectodomains suitable for biochemical, structural, and functional studies. The methodology emphasizes preserving protein structure and function throughout the purification process, which is critical for downstream applications.

How can researchers verify the functionality of expressed TNFRs?

Functional verification of purified TNFRs requires multiple complementary approaches:

Binding kinetics analysis using Surface Plasmon Resonance (SPR):

• Immobilize TNFR1-Fc or TNFR2-Fc on a CM5 sensor chip (target: 3000-3500 RU)

• Prepare serial dilutions of wild-type TNF or TNF mutants (6.25-100 nM)

• Measure association by flowing TNF over immobilized receptors (2 min, 20 μl/min)

• Measure dissociation by flowing buffer (1 min, 20 μl/min)

• Analyze data using global fitting with a 1:1 Langmuir binding model

Cell-based functional assays:

• For TNFR1 activity: Cytotoxicity assay using L-M cells (incubate cells with serial dilutions of TNF for 24h and assess viability using methylene blue assay)

• For TNFR2 activity: Cytotoxicity assay using mTNFR2/mFas-PA cells (incubate with TNF and cycloheximide for 48h and analyze viability using WST-8 assay)

Structural integrity assessment:

• SDS-PAGE under reducing and non-reducing conditions

• Circular dichroism spectroscopy

• Analytical gel filtration to verify oligomeric state

These methodologies provide comprehensive evaluation of both the binding properties and biological activity of the expressed TNFRs, essential for ensuring the reliability of downstream experiments.

How to design TNFR-selective agonists or antagonists?

Developing TNFR-selective modulators requires sophisticated approaches that combine structural knowledge with screening methodologies:

Phage display technique for generating receptor-selective TNF mutants:

• Create a phage library displaying TNF mutants with randomized amino acid residues at the predicted receptor-binding site (nine amino acid residues are typically targeted)

• Perform competitive panning against immobilized TNFR1 and TNFR2

• Isolate phages that bind selectively to the target receptor

• Express and purify selected TNF mutants

• Characterize binding properties and functional activity

This approach has successfully yielded TNFR2-selective agonists with full bioactivity and high receptor specificity, such as the mouse TNFR2-selective TNF mutant described in research findings . Previous attempts using conventional site-directed mutagenesis (e.g., D143N-A145R human TNF mutant) resulted in reduced receptor binding affinity (5-10 fold less than wild-type TNF) .

Evaluation of selectivity and potency:

• SPR analysis to determine binding kinetics to both receptor types

• Competitive binding assays to confirm selectivity

• Cell-based assays using receptor-specific reporter systems

• Cytotoxicity assays on appropriate cell lines

These methodologies enable the development of valuable research tools for dissecting the specific functions of individual TNF receptors in various physiological and pathological contexts.

What techniques are most effective for studying TNFR interactions with other proteins?

Several complementary techniques provide robust data on TNFR interactions with other proteins:

Förster Resonance Energy Transfer (FRET) microscopy:

• Enables visualization of protein interactions in living cells

• For TNFR studies, use ECFP-tagged receptor (donor) and EYFP-tagged binding partner (acceptor)

• Perform acceptor photobleaching FRET experiments

• After photobleaching EYFP-tagged proteins, measure increased emission from ECFP-tagged proteins

• Calculate FRET efficiency as the relative increase in donor fluorescence after acceptor photobleaching

• Statistical analysis of FRET efficiency provides quantitative measure of interaction specificity

Research findings demonstrate high FRET efficiency (approximately 40%) between ECFP-IL-17RD and EYFP-TNFR2, while control pairs (ECFP-IL-17RD/EYFP-TNFR1, ECFP-IL-17RA/EYFP-TNFR2, and ECFP-IL-17RA/EYFP-TNFR1) showed low FRET efficiency (<10%), confirming specific interaction .

Co-immunoprecipitation (Co-IP):

• Detects stable protein-protein interactions in cell lysates

• Can be performed with endogenous proteins or overexpressed tagged versions

• Enables analysis of complex formation in response to stimuli (e.g., TNF-α treatment)

• Western blotting of immunoprecipitates reveals specific interaction partners

• Quantitative analysis can measure relative amounts of interacting proteins

Immunofluorescence co-localization:

• Visualizes spatial overlap of proteins in cells or tissues

• Particularly valuable for tracking receptor trafficking after stimulation

• In renal tubular epithelial cells, IL-17RD and TNFR2 co-localization is enhanced and shifts to perinuclear compartments after TNF-α treatment

• Can be applied to tissue sections for in vivo relevance (e.g., kidney sections from disease models)

How to investigate TNFR signaling pathways in different cellular contexts?

TNFR signaling investigations require context-specific approaches, as signaling outcomes vary significantly between cell types:

Cell type considerations:

• Renal tubular epithelial cells (RTECs) express high levels of TNFR2 but minimal TNFR1

• In RTECs, IL-17RD interacts with TNFR2 and influences NF-κB signaling

• The IL-17RD·TNFR2 complex increases upon TNF-α stimulation

• Different cell types may contain varied co-factors that modulate TNFR signaling

NF-κB activation analysis methodology:

• Transfect cells with NF-κB-responsive luciferase reporter construct

• Treat with appropriate stimuli (TNF-α, receptor-selective agonists)

• Measure luciferase activity as indicator of NF-κB activation

• Include appropriate controls (pathway inhibitors, receptor knockdowns)

• Normalize results to account for transfection efficiency and cell number

Investigation of complex formation dynamics:

• IL-17RD interacts with TNFR2 under basal conditions

• Complex formation increases upon TNF-α stimulation

• Quantitative analysis shows elevated TNFR2 in the complex with IL-17RD after TNF-α treatment

• ELISA experiments confirm enhanced association between IL-17RD-ECD and TNFR2-ECD in the presence of TNF-α

Research findings suggest that the presence of IL-17RD might play a critical role in TNFR2-mediated activation of NF-κB without TNF-α treatment under certain inflammatory conditions, highlighting the complexity of receptor interactions .

How to address common challenges in TNFR expression and purification?

Researchers frequently encounter several challenges when working with TNFRs in Sf9 expression systems. Methodological solutions include:

For protein verification after purification:

• Analyze protein homogeneity via analytical size exclusion chromatography

• Confirm molecular weight via mass spectrometry

• Verify binding activity using surface plasmon resonance

• Assess thermal stability using differential scanning fluorimetry

These methodological approaches help overcome common technical hurdles in TNFR research and ensure the production of high-quality proteins for downstream applications.

What controls are essential for TNFR interaction studies?

Rigorous experimental controls are critical for obtaining reliable data in TNFR interaction studies:

FRET experiments:

• Negative controls: Measure FRET efficiency between unrelated proteins (e.g., ECFP-IL-17RA and EYFP-TNFR1) to establish background levels (<10%)

• Positive controls: Use known interacting protein pairs

• Expression level controls: Normalize for protein expression levels

• Proper statistical analysis of FRET efficiency data across multiple experiments

Co-immunoprecipitation studies:

• Input controls: Analyze total lysate to verify expression of target proteins

• Negative controls: Use isotype-matched irrelevant antibodies for immunoprecipitation

• Reciprocal co-IPs: Perform experiments immunoprecipitating each protein partner

• Specificity controls: Include competing peptides or proteins when possible

Stimulus-response experiments:

• Time course: Analyze complex formation at multiple time points after stimulation

• Dose-response: Test multiple concentrations of stimulus (e.g., TNF-α)

• Pathway inhibitors: Include specific inhibitors to verify signaling mechanisms

• Receptor mutants: Use binding-deficient mutants to confirm specificity

How to interpret seemingly contradictory findings in TNFR research?

TNFR biology is complex, and apparent contradictions in research findings often reflect context-dependent functioning rather than actual inconsistencies. Methodological approaches to resolving these challenges include:

Context-dependent TNFR2 signaling assessment:

• TNFR2 signaling often requires co-factors that vary between cell types

• In RTECs, IL-17RD serves as a co-receptor for TNFR2, enabling NF-κB activation

• IL-17RD's membrane-bound status affects its role in NF-κB signaling (the Y330F membrane-bound mutant abrogates inhibitory function)

• TNFR2 can function in both TNF-α-dependent and -independent manners

Species-specific considerations:

• Human and mouse TNFRs show structural and functional differences

• Human TNF cannot selectively stimulate mouse TNFR2

• Species-specific TNFR-selective TNF mutants may be required for certain studies

• Previous human TNFR2-selective TNF mutant (R2-7) does not bind to mouse TNFR2

Comparing experimental systems:

• Cell types: Different cells express varying levels of TNFRs and co-receptors

• Receptor expression levels: Analyze whether studies used endogenous or overexpressed receptors

• Stimulation conditions: Compare acute vs. chronic TNF exposure

• Readout methods: Different assays may capture distinct aspects of receptor function

When evaluating contradictory findings, researchers should systematically compare experimental conditions, examine receptor expression patterns, and consider the presence of potential co-factors that might explain the observed differences.