Recombinant Macaca mulatta Tumor necrosis factor protein (TNF), partial, (Active)

What is TNF-alpha and what are its primary functions in the rhesus macaque immune system?

TNF-alpha, also known as cachectin and TNFSF2, is a pleiotropic molecule that plays central roles in inflammation, immune system development, apoptosis, and lipid metabolism . In rhesus macaques, TNF-alpha consists of a 35 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 177 amino acid extracellular domain (ECD) . It functions as a key mediator of inflammatory responses, similar to human TNF-alpha.

TNF-alpha is produced by various immune cells (macrophages, monocytes, T cells, B cells), as well as epithelial cells, endothelial cells, and tumor cells . The protein is assembled intracellularly to form a noncovalently linked homotrimer expressed on the cell surface, which can induce lysis of neighboring tumor cells and virus-infected cells . Membrane-bound TNF-alpha can be cleaved by TACE/ADAM17 to release a bioactive soluble cytokine .

The biological effects of TNF-alpha are mediated through two receptors: the ubiquitous 55-60 kDa TNF RI, which contains a cytoplasmic death domain triggering apoptosis, and the hematopoietic cell-restricted 80 kDa TNF RII . Both receptors bind TNF-alpha with comparable affinity .

How similar is rhesus macaque TNF-alpha to human TNF-alpha?

Rhesus macaque TNF-alpha shares remarkable homology with human TNF-alpha, making it valuable for translational research. Within the extracellular domain, rhesus TNF-alpha shares 97% amino acid sequence identity with human TNF-alpha . This high conservation extends to the structural organization of the protein, with both species having similar domain structures.

Despite this similarity in protein sequence, there are important differences in the regulatory regions. Polymorphisms in the human TNF-alpha promoter known to be associated with malaria susceptibility are not shared with macaques . Sequence analysis of the TNF-alpha promoter region in macaques revealed a total of 14 single nucleotide polymorphisms (SNPs), with 20 unique haplotypes identified across different macaque populations .

What is the molecular structure of recombinant rhesus macaque TNF-alpha?

Recombinant rhesus macaque TNF-alpha is typically produced as the soluble extracellular domain portion of the native protein, spanning from Val77 to Leu233 . Based on comparative analysis with human TNF-alpha, we can infer that rhesus TNF-alpha forms a homotrimer with a molecular weight of approximately 53 kDa in its native state .

When analyzed by SDS-PAGE under reducing conditions, the monomeric form appears as a band at approximately 17 kDa . The trimeric structure is critical for biological activity, as it enables proper receptor binding and signaling.

E. coli-derived rhesus macaque TNF-alpha is commonly used in research applications . The recombinant protein can be produced either with a carrier protein (typically bovine serum albumin, BSA) to enhance stability or in carrier-free formulations for applications where BSA might interfere .

How should I design experiments to evaluate the biological activity of recombinant rhesus macaque TNF-alpha?

Evaluating the biological activity of recombinant rhesus macaque TNF-alpha requires well-designed assays that capture its key functions. The most established method is the L-929 mouse fibroblast cytotoxicity assay in the presence of actinomycin D. The ED50 (effective dose for 50% cytotoxicity) for biologically active rhesus macaque TNF-alpha should be in the range of 15-60 pg/mL .

For this assay:

• Seed L-929 cells in 96-well plates

• Add actinomycin D (typically 1 μg/mL)

• Add serial dilutions of TNF-alpha

• Assess cell viability after 18-24 hours

• Calculate ED50 values

Additional assays to consider include:

• NF-κB activation assays: Using reporter cell lines expressing luciferase under NF-κB responsive elements to measure signaling pathway activation

• Proliferation assays: Recombinant TNF-alpha can enhance proliferation of certain cell types, such as rhesus B cell lines

• Cytokine induction: Measure the production of downstream inflammatory cytokines (IL-1β, IL-6, IL-8) in response to TNF-alpha stimulation

• Western blotting: To detect phosphorylation of signaling molecules in the TNF pathway (p38 MAPK, JNK, IKK)

Always include appropriate controls:

• Positive controls: Human TNF-alpha

• Negative controls: Heat-inactivated TNF-alpha, unrelated proteins

• Specificity controls: TNF-alpha neutralizing antibodies

How do genetic polymorphisms in the TNF-alpha promoter region affect expression in rhesus macaques?

Genetic polymorphisms in the TNF-alpha promoter region can significantly impact TNF-alpha expression in rhesus macaques, potentially influencing disease susceptibility and experimental outcomes. A study examining 40 macaques, including M. mulatta of Chinese and Indian ancestry and M. fascicularis, identified important genetic variations .

The analysis revealed 14 single nucleotide polymorphisms (SNPs) in the TNF-alpha promoter region, five of which were newly described at the time . These polymorphisms can potentially influence transcription factor binding, affecting regulation of TNF-alpha expression. The TFSEARCH program was used to investigate the potential of these polymorphisms to influence transcription factor binding .

These genetic variations could contribute to differential disease susceptibility observed among different macaque populations. Researchers should consider the genetic background of animals used in their studies, as differences in TNF-alpha promoter polymorphisms could affect experimental results and their interpretation.

What are the optimal storage and handling conditions for recombinant rhesus TNF-alpha to maintain bioactivity?

Proper storage and handling of recombinant rhesus macaque TNF-alpha is critical for maintaining its biological activity. Based on product information, the following conditions are recommended:

For lyophilized protein:

• Store at -20°C to -80°C upon receipt

• The product is typically shipped at ambient temperature

Reconstitution protocols:

• For preparations with BSA carrier: Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For carrier-free preparations: Reconstitute at 100 μg/mL in sterile PBS without additives

Storage after reconstitution:

• Prepare aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C to -80°C

• Use a manual defrost freezer to avoid temperature fluctuations

• Avoid exposure to strong light and oxidizing agents

Handling precautions:

• Thaw aliquots on ice or at 2-8°C, never at room temperature

• Once thawed, use immediately or store at 2-8°C for short-term (1-2 weeks)

• Prepare working dilutions on ice and use within the same day

The presence of carrier proteins like BSA enhances protein stability and increases shelf-life . For applications where BSA could interfere (such as certain immunoassays), use carrier-free preparations.

What analytical methods are most appropriate for characterizing recombinant rhesus macaque TNF-alpha?

Comprehensive characterization of recombinant rhesus macaque TNF-alpha requires multiple analytical approaches to assess identity, purity, structure, and biological activity:

Physicochemical Characterization:

• SDS-PAGE Analysis: Under reducing conditions, monomeric TNF-alpha appears as a band at approximately 17 kDa . Silver staining provides higher sensitivity for detecting impurities.

• Size Exclusion Chromatography (SEC): For analyzing the oligomeric state (homotrimer) and aggregation status. SEC coupled with multi-angle light scattering (SEC-MALS) can determine absolute molecular weight, which should be approximately 53 kDa for the trimeric form .

• Mass Spectrometry: For precise molecular weight determination and primary structure confirmation.

Immunological Characterization:

• ELISA: For quantitative determination of concentration.

• Western Blotting: For identity confirmation using specific antibodies.

• Surface Plasmon Resonance (SPR): For measuring binding kinetics to TNF receptors.

Biological Activity Assessment:

• Cytotoxicity Assay: The L-929 mouse fibroblast cytotoxicity assay with actinomycin D. The ED50 should be in the range of 15-60 pg/mL .

• Cell-Based Reporter Assays: Such as NF-κB reporter assays to measure activation of downstream signaling.

• Proliferation Assays: To assess the ability to enhance B cell proliferation .

Contaminant Analysis:

• Endotoxin Testing: Using Limulus Amebocyte Lysate (LAL) assay, critical for E. coli-derived proteins.

• Host Cell Protein (HCP) Analysis: Using ELISA or mass spectrometry-based methods.

By combining these methods, researchers can thoroughly characterize recombinant rhesus macaque TNF-alpha for research applications.

How can I determine the appropriate concentration of recombinant rhesus macaque TNF-alpha for my specific assay?

Determining the optimal concentration of recombinant rhesus macaque TNF-alpha requires systematic titration based on assay type:

Step 1: Literature-based starting point
From published data, we know that for cytotoxicity assays in L-929 cells with actinomycin D, the ED50 is 15-60 pg/mL . This provides a reference range for initial testing.

Step 2: Broad-range dose-response experiment

• Prepare serial dilutions spanning 5-6 log concentrations (e.g., 0.1 pg/mL to 10 μg/mL)

• Include vehicle control (0 pg/mL)

• Assess biological response using appropriate readouts

• Plot the dose-response curve and identify:

  • Minimum effective concentration

  • ED50 (concentration producing 50% of maximum response)

  • Saturation concentration

Step 3: Refined titration
Narrow the concentration range around the effective zone identified in Step 2, using smaller increments.

Concentration guidelines for different assay types:

Additional considerations:

• Time-dependence: Test multiple time points to capture optimal response

• Cell density: Optimize cell numbers for each assay type

• Medium composition: Serum can contain TNF binding proteins

• Species specificity: Rhesus TNF-alpha may have different potency on cells from different species

This systematic approach will help determine the appropriate concentration range specific to your experimental system.

What are the best approaches for detecting TNF-alpha expression in rhesus macaque tissue samples?

Detecting TNF-alpha expression in rhesus macaque tissue samples requires methods that are both sensitive and specific:

RNA-Based Detection Methods:

RT-PCR and qRT-PCR:

• Design primers specific to rhesus macaque TNF-alpha sequence

• Target regions that differ from closely related species

• Include appropriate housekeeping genes for normalization

• Provides quantitative assessment of TNF-alpha mRNA levels

RNA In Situ Hybridization (RNA-ISH):

• Allows visualization of TNF-alpha mRNA within tissue context

• Preserves spatial information about cell types expressing TNF-alpha

• Can be combined with immunohistochemistry for cell type identification

Protein-Based Detection Methods:

ELISA:

• For quantitative measurement of TNF-alpha protein in tissue homogenates

• Use commercially available kits validated for rhesus macaque samples

• Typical detection limit: 1-10 pg/mL

Western Blotting:

• Can distinguish between membrane-bound (26 kDa) and soluble (17 kDa) forms

• Requires validation of antibody specificity for rhesus macaque TNF-alpha

Immunohistochemistry (IHC):

• Preserves tissue architecture and cellular context

• Use antibodies validated for rhesus macaque TNF-alpha

• Consider antigen retrieval methods to improve detection

Flow Cytometry:

• For detecting TNF-alpha in single-cell suspensions

• Particularly useful for identifying specific cell populations producing TNF-alpha

• Requires tissue disaggregation and intracellular staining protocols

Optimization considerations:

Combining multiple detection methods provides the most comprehensive assessment of TNF-alpha expression in tissue samples.

What controls should be included when using recombinant rhesus macaque TNF-alpha in experimental systems?

When designing experiments with recombinant rhesus macaque TNF-alpha, proper controls are essential for valid interpretation of results:

For in vitro studies:

Negative Controls:

• Vehicle Control: The same buffer used for TNF-alpha dilution (e.g., PBS with carrier protein)

• Heat-Inactivated TNF-alpha: TNF-alpha that has been heat-denatured (e.g., 95°C for 10 minutes)

• Unrelated Recombinant Protein: Another recombinant protein of similar size prepared using the same expression system

• Unstimulated Cells: Cells maintained in culture media without any treatment

Positive Controls:

• Human TNF-alpha: Given the 97% sequence identity, this can serve as a reference standard

• Known TNF-alpha Inducers: For instance, LPS for macrophage activation

• Established Cell Lines: Cell lines with well-characterized responses to TNF-alpha

Specificity Controls:

• TNF-alpha Neutralizing Antibodies: To confirm that observed effects are specifically due to TNF-alpha

• TNF Receptor Antagonists: To confirm receptor-mediated effects

• Dose-Response: Multiple concentrations of TNF-alpha to establish relationship between dose and response

For in vivo studies:

Study Design Controls:

• Vehicle Treatment: Animals receiving the same vehicle used for TNF-alpha administration

• Irrelevant Protein Control: Animals receiving an unrelated recombinant protein

• TNF-alpha Blockade: Co-administration of TNF-alpha with neutralizing antibodies

• Dose-Response Studies: Multiple dosage groups

• Time-Course Studies: Sampling at multiple time points

Incorporating these controls helps attribute observed effects specifically to recombinant rhesus macaque TNF-alpha and distinguishes them from experimental artifacts or non-specific effects.

How does the presence of carrier protein (BSA) affect experimental outcomes when using recombinant rhesus macaque TNF-alpha?

The presence of bovine serum albumin (BSA) as a carrier protein in recombinant rhesus macaque TNF-alpha preparations can significantly impact experimental outcomes:

Benefits of carrier protein:

• Enhances protein stability by preventing adsorption to surfaces

• Increases shelf-life by protecting against degradation

• Allows the recombinant protein to be stored at more dilute concentrations

• Maintains biological activity during freeze-thaw cycles

Potential experimental implications:

• Immunological assays: BSA can interfere with antibody-based detection methods, especially when:

  • Using anti-bovine antibodies that might cross-react with BSA

  • Developing immunoassays where BSA might compete for binding

  • Performing immunoprecipitation experiments

• Cell culture applications:

  • BSA may contain bioactive contaminants that affect cellular responses

  • For serum-free culture systems, BSA might introduce undefined factors

  • In low-serum conditions, carrier BSA might affect baseline cellular functions

• Protein interaction studies:

  • BSA can non-specifically bind to other proteins

  • May interfere with protein-protein interaction studies

  • Could affect surface plasmon resonance or other binding assays

When to use carrier-free preparations:
The carrier-free version (without BSA) is recommended for applications in which the presence of BSA could interfere , including:

• Mass spectrometry-based analyses

• Crystallization studies

• Certain receptor binding assays

• Development of diagnostic tests

• Experiments requiring precise protein quantification

Control strategies:

• Include BSA-only controls at equivalent concentrations

• Compare results between carrier-containing and carrier-free preparations

• Include appropriate blocking steps in immunoassays to minimize BSA interference

• Consider the final BSA concentration in the experimental system

By understanding these considerations, researchers can select the appropriate TNF-alpha preparation format and implement necessary controls to ensure valid experimental outcomes.

How can I validate the specificity of biological responses to recombinant rhesus macaque TNF-alpha?

Validating the specificity of biological responses to recombinant rhesus macaque TNF-alpha is crucial for ensuring reliable and interpretable results:

Receptor Blocking Experiments:

• Use neutralizing antibodies against TNF receptors (TNFR1 and TNFR2)

• Pretreat cells with these antibodies before adding recombinant TNF-alpha

• A significant reduction in TNF-alpha effects confirms receptor-specific action

• Include isotype control antibodies as negative controls

TNF-alpha Neutralization:

• Use anti-TNF-alpha neutralizing antibodies

• Preincubate recombinant TNF-alpha with these antibodies before addition to cells

• Abolishment of the biological effect confirms specificity

• Include non-specific antibodies as controls

Dose-Response Relationships:

• Perform detailed dose-response experiments

• Plot the relationship between TNF-alpha concentration and biological response

• A sigmoidal dose-response curve with EC50 values in the expected range (pg/mL to ng/mL) supports specificity

• Unusual dose-response patterns may indicate non-specific effects

Pathway-Specific Inhibitors:

• Use inhibitors of TNF signaling pathways (e.g., NF-κB inhibitors)

• If these block the effects of recombinant TNF-alpha, this supports pathway-specific action

• Include appropriate vehicle controls

Cross-Species Validation:

• Compare the effects of rhesus macaque TNF-alpha with human TNF-alpha

• Similar patterns of biological activity but potentially different potencies would be expected based on the 97% sequence identity

• Include species-specific controls when possible

Biological Activity Profile:

• Test multiple biological activities associated with TNF-alpha (cytotoxicity, cytokine induction, NF-κB activation)

• A consistent pattern of activity across different assays supports specificity

• Include positive and negative controls in each assay

By employing multiple validation approaches, researchers can confidently establish the specificity of biological responses to recombinant rhesus macaque TNF-alpha.

How can recombinant rhesus macaque TNF-alpha be used in infectious disease research models?

Recombinant rhesus macaque TNF-alpha serves as a valuable tool in infectious disease research, particularly for studies using non-human primates as models for human diseases:

HIV/SIV Research:

• Study TNF-alpha's role in immune activation during chronic infection

• Investigate interactions between viral proteins and TNF signaling pathways

• Evaluate TNF-alpha as a biomarker for disease progression

• Test anti-TNF strategies as adjunctive therapies

Tuberculosis Models:

• Examine TNF-alpha's role in granuloma formation and maintenance

• Study the impact of TNF blockade on TB reactivation

• Investigate host-directed therapies targeting TNF signaling

• Assess TNF-alpha as a biomarker for treatment response

Malaria Research:

• Investigate the role of TNF-alpha in pathogenesis of severe malaria

• Study the relationship between TNF-alpha polymorphisms and disease susceptibility

• Test anti-TNF interventions to reduce immunopathology

• Examine TNF-alpha's role in developing protective immunity

Viral Hemorrhagic Fevers:

• Study TNF-alpha's contribution to vascular leak syndrome

• Evaluate TNF-alpha as a biomarker for disease severity

• Test anti-TNF strategies to reduce immunopathology

• Investigate cytokine networks during infection

Experimental Approaches:

• Ex vivo stimulation: Use recombinant TNF-alpha to stimulate immune cells from infected animals

• In vivo administration: Administer TNF-alpha to study systemic effects

• Neutralization studies: Use anti-TNF antibodies to block endogenous TNF-alpha

• Co-culture systems: Study interactions between infected cells and TNF-alpha-producing cells

The 97% sequence identity between rhesus and human TNF-alpha makes rhesus macaque models particularly valuable for translational research on infectious diseases that are difficult to study directly in humans.

What are the critical factors to consider when using recombinant rhesus macaque TNF-alpha in inflammation and autoimmunity studies?

When using recombinant rhesus macaque TNF-alpha in inflammation and autoimmunity studies, several critical factors must be considered to ensure valid and translatable results:

Dosing and Pharmacokinetics:

• Determine physiologically relevant doses based on measured levels in disease states

• Consider the half-life of recombinant TNF-alpha in experimental systems

• Develop appropriate dosing schedules for chronic versus acute inflammation models

• Account for differences in local versus systemic administration

Receptor Specificity and Distribution:

• Consider the differential expression of TNFR1 (ubiquitous) versus TNFR2 (hematopoietic-restricted)

• Account for potential differences in receptor distribution between tissues

• Remember that only TNFR1 contains a cytoplasmic death domain triggering apoptosis

• Consider the impact of soluble TNF receptors that can neutralize TNF-alpha activity

Genetic Background Considerations:

• Be aware of TNF-alpha promoter polymorphisms in different macaque populations

• Consider how genetic background might influence TNF-alpha responsiveness

• Ensure consistent genetic backgrounds within experimental groups

• Account for potential differences between Chinese-origin versus Indian-origin rhesus macaques

Model-Specific Factors:

• For colitis models: consider TNF-alpha's effects on intestinal barrier function

• For arthritis models: account for TNF-alpha's effects on synoviocytes and osteoclasts

• For psoriasis models: consider TNF-alpha's impact on keratinocyte proliferation

• For multiple sclerosis models: account for TNF-alpha's dual roles in CNS inflammation

Experimental Design Considerations:

• Include appropriate timing for TNF-alpha administration relative to disease induction

• Consider combination treatment with other cytokines that synergize with TNF-alpha

• Account for potential compensatory mechanisms with chronic TNF-alpha exposure

• Include adequate controls for carrier proteins and endotoxin contamination

Translational Relevance:

• Compare findings to human studies whenever possible

• Consider the impact of anatomical and physiological differences between species

• Evaluate how differences in immune system development might affect outcomes

• Account for differences in environmental exposures between laboratory animals and humans

By carefully considering these factors, researchers can maximize the translational value of studies using recombinant rhesus macaque TNF-alpha in inflammation and autoimmunity research.