Recombinant Human Tumor necrosis factor protein (TNF), partial (Active)

What is the molecular structure of recombinant human TNF-alpha?

Recombinant human TNF-alpha is typically expressed as an E. coli-derived protein spanning Val77-Leu233 of the native sequence, with or without an N-terminal Met residue. The functional protein exists as a homotrimer with a molecular weight of approximately 53.1 kDa as determined by SEC-MALS analysis, although the monomeric form appears as a 17 kDa band under reducing SDS-PAGE conditions . The complete native human TNF-alpha consists of a 35 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 177 amino acid extracellular domain (ECD) . For research purposes, it's critical to note that the recombinant form represents the soluble, cleaved portion of the protein that corresponds to the bioactive extracellular domain.

How does recombinant human TNF-alpha compare structurally with TNF-alpha from other species?

Within the extracellular domain, human TNF-alpha shares 97% amino acid sequence identity with rhesus macaque TNF-alpha and between 71-92% identity with TNF-alpha from bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat sources . This high degree of conservation reflects the evolutionary importance of this cytokine. When designing cross-species experiments, researchers should account for these homology differences, particularly when evaluating receptor binding specificity or developing neutralizing antibodies.

What is the standard method for measuring TNF-alpha bioactivity?

The standard bioassay for TNF-alpha activity utilizes the L-929 mouse fibroblast cytotoxicity assay in the presence of actinomycin D (a metabolic inhibitor). The effective dose (ED50) typically ranges from 25-100 pg/mL . This assay measures the cytotoxic potential of TNF-alpha and serves as the gold standard for activity determination. Alternative methods include receptor binding assays, reporter gene assays using NF-κB responsive elements, and measurement of specific downstream signaling events such as phosphorylation of IκB.

What are the optimal storage and handling conditions for maintaining recombinant TNF-alpha stability?

Lyophilized recombinant TNF-alpha should be stored at -20°C to -80°C. After reconstitution in sterile PBS containing at least 0.1% carrier protein (such as BSA), the protein remains stable for up to one month at 2-8°C or can be aliquoted and stored at -20°C to -80°C for up to 3 months with minimal loss of activity. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and activity loss. Working solutions should be prepared fresh on the day of experimentation whenever possible, and sterile technique should be employed to prevent microbial contamination.

How should TNF-alpha dose-response experiments be designed for cell-based assays?

When designing dose-response experiments with TNF-alpha, researchers should:

• Establish a concentration range spanning at least 5 orders of magnitude (e.g., 0.1 pg/mL to 10 ng/mL)

• Include a positive control (e.g., validated active TNF-alpha)

• Determine cell type-specific responses, as sensitivity varies widely between cell types

• Consider temporal dynamics, with typical response times ranging from 4-24 hours for transcriptional changes

• Account for potential synergistic effects with other cytokines or stimuli

For cytotoxicity assays, the L-929 cell line with actinomycin D demonstrates optimal sensitivity with an ED50 of 25-100 pg/mL . For NF-κB activation assays in 293T cells, dose-response curves typically show activity in the nanomolar range, with compounds like S10 and 4e demonstrating IC50 values of 14 μM and 3 μM, respectively, when inhibiting TNF-induced activity .

What methodological approaches can be used to study TNF-alpha receptor binding?

Researchers can employ several complementary techniques to study TNF-alpha receptor binding:

• Surface Plasmon Resonance (SPR): Allows real-time measurement of binding kinetics between immobilized TNFR and flowing TNF-alpha. This technique can detect competitive binding, as demonstrated in studies of small molecule inhibitors that block TNF-α and TNFR interactions .

• Fluorescence Resonance Energy Transfer (FRET): Enables visualization of receptor-ligand interactions in living cells.

• Radioligand binding assays: Provides quantitative measurement of binding affinity and receptor density.

• Molecular modeling and docking: The crystal structure of TNF-α dimer (PDB code: 2AZ5) can be used for in silico prediction of binding interactions .

• Cross-linking studies: Identifies specific binding sites and interaction domains.

Each method offers distinct advantages, and combining multiple approaches provides more robust characterization of receptor-ligand interactions.

How can recombinant TNF-alpha be used to investigate endothelial activation and procoagulant activity?

Recombinant TNF-alpha directly activates human vascular endothelial cells to induce tissue factor-like procoagulant activity (PCA). Methodologically, this can be studied through:

• Isolation of primary endothelial cells from umbilical veins, saphenous veins, iliac arteries, or thoracic aortae

• Treatment with rTNF at concentrations ranging from 100-500 units/ml

• Measurement of PCA using one-stage clotting assays after 4-hour incubation

• Assessment of PCA expression on intact viable endothelial monolayers

The response kinetics typically show a rapid rise to peak activity at approximately 4 hours, followed by a decline toward basal levels by 24 hours . This experimental system offers insights into thrombotic complications associated with inflammatory conditions. Importantly, the effects of TNF can be distinguished from those of IL-1 using specific neutralizing antisera, though their effects may be additive even at apparent maximal doses .

What are the pharmacokinetic parameters of recombinant TNF-alpha in vivo?

Clinical pharmacology studies with recombinant human TNF in cancer patients have established the following pharmacokinetic parameters:

The rapid clearance suggests that continuous infusion or frequent dosing may be necessary to maintain therapeutic levels in experimental models . For research design, these parameters should inform dosing schedules and sampling timepoints, particularly in pharmacodynamic studies.

What are the most effective approaches for developing small molecule inhibitors targeting TNF-alpha?

Development of small molecule TNF-alpha inhibitors can follow this methodological framework:

• Structure-based design utilizing the crystal structure of TNF-α dimer (PDB code: 2AZ5)

• Shape screening of compound libraries using established inhibitors as templates

• Rational design of derivatives based on structure-activity relationships

• Combined in silico and experimental validation through:

  • SPR competitive binding assays between TNF-α and TNFR

  • Cell-based assays measuring NF-κB activation

  • Analysis of structural interactions using molecular docking

This approach has yielded compounds like 4e, a 2-oxo-N-phenyl-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivative with an IC50 of 3.0 ± 0.8 μM in cell-based assays, representing one of the most potent TNF-α small molecule inhibitors reported . Researchers should focus on optimizing both binding affinity and pharmacokinetic properties when developing novel inhibitors.

What adverse effects should researchers consider when designing TNF-alpha experiments?

When designing experiments involving TNF-alpha administration, researchers should anticipate potential adverse effects based on clinical observations:

• Constitutional symptoms: fever, chills, headache, fatigue

• Cardiovascular effects: hypotension requiring fluid administration

• Hematological changes: acute alterations in granulocyte, lymphocyte, and monocyte counts

• Metabolic responses: changes in serum zinc levels and plasma cortisol consistent with acute phase response

• Local inflammation: severe reactions at subcutaneous injection sites, particularly at doses of 125-250 μg/m²

• Hemostatic alterations: minor changes in parameters related to coagulation

These effects should inform experimental design, particularly for in vivo studies, where appropriate monitoring and supportive measures should be planned . Researchers should implement the principle of refinement in animal studies by incorporating these considerations into humane endpoints and monitoring protocols.

How can researchers analyze TNF-alpha inhibitor safety profiles using pharmacovigilance data?

Analysis of TNF-alpha inhibitor safety can be approached using disproportionality analysis of pharmacovigilance databases such as the FDA Adverse Event Reporting System. The recommended statistical methods include:

• Reporting Odds Ratio (ROR) with 95% confidence intervals

• Information Component (IC) using Bayesian Confidence Propagation Neural Network

Signal detection criteria:

• ROR025 (lower limit of 95% CI) > 1 and adverse event count > 3

• IC025 (lower limit of 95% credible interval) > 0

Sensitivity analyses should include sex stratification to evaluate gender-specific signals . Visualization techniques such as UpSet plots can help identify common adverse events across different TNF inhibitors versus those unique to specific agents. This methodological approach provides systematic evidence for safety signal detection beyond anecdotal reports.

How can researchers address TNF-alpha experimental variability across different cell types?

Cell type-specific responses to TNF-alpha represent a significant source of experimental variability. To address this:

• Characterize baseline expression levels of TNF receptors (TNFR1 and TNFR2) in your cell system using flow cytometry or Western blotting

• Determine the relative contributions of each receptor using selective blocking antibodies

• Assess the presence of soluble TNF receptors in your culture system that might neutralize TNF activity

• Establish cell type-specific dose-response curves rather than relying on published ED50 values

• Consider potential synergistic or antagonistic effects with other cytokines present in your experimental system

For primary cells, passage number and donor variability should be documented and accounted for in experimental design. Standardization of culture conditions (serum concentration, cell density, substrate) is essential for reproducible TNF-alpha responses.

What are the critical considerations when combining TNF-alpha with other cytokines or therapeutic agents?

When designing combination experiments with TNF-alpha, researchers should:

• Determine sequence-dependent effects: the order of administration can significantly affect outcomes

• Establish appropriate dose ratios based on the relative potencies of each agent

• Account for potential receptor cross-talk and shared downstream signaling pathways

• Consider temporal aspects of signaling dynamics for each agent

• Validate additive, synergistic, or antagonistic effects using appropriate mathematical models (e.g., Chou-Talalay method)

Clinical studies suggest that combinations of TNF-alpha with other cytokines and cytostatic agents may produce enhanced therapeutic effects compared to TNF-alpha monotherapy . This principle likely extends to experimental systems, necessitating careful experimental design to distinguish mechanistic interactions from simple additive effects.

What are the emerging approaches for studying TNF-alpha structure-function relationships?

Advanced structural biology techniques are expanding our understanding of TNF-alpha's molecular mechanism:

• Cryo-electron microscopy to visualize TNF-receptor complexes in near-native states

• Hydrogen-deuterium exchange mass spectrometry to map dynamic protein interactions

• Single-molecule imaging to track TNF-receptor clustering and signaling complex formation

• Structural proteomics to identify post-translational modifications affecting activity

• Computational molecular dynamics simulations to model conformational changes during receptor engagement

These approaches can help resolve outstanding questions about how TNF-alpha trimers engage with receptor complexes and how this leads to differential signaling outcomes. The application of these techniques could inform the development of more selective TNF modulators with improved therapeutic profiles.

How can genetic and pharmacological perturbation be combined to study TNF-alpha signaling networks?

An integrated approach combining genetic and pharmacological tools provides powerful insights into TNF-alpha biology:

• CRISPR-Cas9 knockout/knockin models of specific pathway components

• Small molecule inhibitors with defined mechanisms targeting discrete pathway nodes

• Phosphoproteomic analysis to map signaling cascades with temporal resolution

• Single-cell transcriptomics to capture population heterogeneity in responses

• Optogenetic or chemically-inducible systems for precise temporal control of TNF signaling

This multi-modal approach can help deconvolute the complex signaling networks downstream of TNF-alpha receptors and identify critical nodes that might serve as therapeutic targets. Importantly, this strategy can reveal compensatory mechanisms that may limit the efficacy of TNF inhibitors in certain contexts.