Recombinant Rhesus Macaque Interferon gamma protein (IFNG) (Active)

What is the amino acid sequence of recombinant rhesus macaque interferon gamma protein?

Recombinant rhesus macaque interferon gamma protein typically consists of amino acids Gln24-Gln165, with an N-terminal Met when expressed in E. coli expression systems. The full sequence includes: QDPYVKEAENLKKYFNAGDPDVADNGTLFLGILRNWKEESDRKIMQSQIVSFYFKLFKNFKDDQRIQKSVETIKEDINVKFFNSNKKKRDDFEKLTNYSVIDSNVQRKAVHELIQVMAELSPAAKIGKRKRSQMFRGRRASQ . This sequence composition is critical for its biological activity and structural integrity in experimental systems.

How does rhesus macaque IFNG differ structurally from human IFNG?

Rhesus macaque IFNG shares high sequence homology with human IFNG, reflecting their evolutionary relationship. The core functional domains remain conserved between species, though specific amino acid differences exist that may affect receptor binding affinity and downstream signaling efficiency. These subtle structural variations must be considered when translating experimental findings between species. Phylogenetic analysis demonstrates that macaque IFNG is highly conserved among primates, making it a valuable model for human IFNG function in certain experimental contexts .

What are the optimal storage conditions for maintaining IFNG activity?

For maximum stability and biological activity retention, recombinant rhesus macaque IFNG should be stored in a manual defrost freezer, avoiding repeated freeze-thaw cycles that can compromise protein integrity. The lyophilized form provides greater stability for long-term storage. Once reconstituted, the protein should be stored at -20°C to -80°C in small aliquots to prevent repeated freeze-thaw cycles. Carrier-containing formulations (with BSA) generally offer enhanced stability during storage compared to carrier-free versions .

What are the primary signaling pathways activated by rhesus macaque IFNG?

Rhesus macaque IFNG primarily activates the JAK-STAT signaling pathway. Upon binding to its receptor IFNGR1, the receptor's intracellular domain undergoes conformational changes that allow association of downstream signaling components JAK2, JAK1, and STAT1. This interaction leads to STAT1 phosphorylation, dimerization, nuclear translocation, and subsequent transcription of IFNG-regulated genes . Secondary pathways may include MAPK, PI3K, and NF-κB signaling cascades that contribute to the diverse biological effects of IFNG in immune regulation.

How does IFNG enhance antigen presentation in macaque immune cells?

IFNG enhances antigen presentation through multiple mechanisms: (1) It induces replacement of constitutive catalytic proteasome subunits with immunoproteasome subunits, increasing the quantity, quality, and repertoire of peptides available for class I MHC loading; (2) It upregulates expression of activator PA28, which associates with the proteasome and alters proteolytic cleavage preferences; (3) It enhances MHC class II expression on cell surfaces by promoting expression of several key molecules including cathepsins B, H, and L; (4) It increases expression of transporters associated with antigen processing (TAP1/2) . These coordinated actions collectively amplify the cell's capacity to process and present antigens to T cells.

How do carrier-free versus carrier-containing IFNG formulations affect biological activity?

Carrier-free and carrier-containing formulations of recombinant rhesus macaque IFNG exhibit different properties that can impact experimental outcomes:

The choice between formulations should be guided by the specific experimental requirements, with carrier-free versions preferred for applications where the presence of BSA could potentially interfere with results .

How can recombinant rhesus macaque IFNG be used to model SIV/HIV immune responses?

Recombinant rhesus macaque IFNG serves as a valuable tool for studying immune responses in SIV/HIV research due to the rhesus macaque's status as a premier model for human immunodeficiency virus infections. Methodologically, researchers can:

• Use recombinant IFNG to stimulate macaque PBMCs ex vivo to assess immune cell activation and antiviral responses

• Measure endogenous IFNG production in response to SIV antigens as a metric of T cell functionality

• Compare IFNG-induced gene expression profiles between uninfected and SIV-infected macaques to identify disrupted immune pathways

• Evaluate the efficacy of IFNG as an immunotherapeutic adjuvant in vaccine development studies

• Investigate the interplay between IFNG and mucosal immunity, particularly relevant as mucosal tissues are primary sites for viral transmission

These applications provide insights into how interferons modulate immune responses during primate lentiviral infections and can inform human HIV therapeutic strategies.

What are appropriate positive controls when testing recombinant rhesus macaque IFNG bioactivity?

When assessing the bioactivity of recombinant rhesus macaque IFNG, several positive control approaches are recommended:

• Induction of MHC class II expression on macaque monocytes/macrophages (flow cytometry)

• Stimulation of JAK-STAT pathway activation measured by STAT1 phosphorylation (Western blot or flow cytometry)

• Upregulation of interferon-stimulated genes (ISGs) such as IRF1, CXCL10, or IDO1 (qPCR)

• Enhancement of antiviral activity against VSV or EMCV in rhesus macaque fibroblasts (viral inhibition assay)

• Induction of nitric oxide production in macaque macrophages (Griess assay)

The ED50 for many of these effects typically ranges from 50-500 ng/mL, depending on the specific assay and cellular system . Incorporating dose-response curves with these readouts provides robust validation of protein activity.

How can recombinant IFNG be used to study differences in immune responses between macaque species?

Recombinant IFNG provides a powerful tool for comparative immunology studies across different macaque species, which can reveal evolutionary adaptations to pathogen pressures:

• Compare IFNG-induced gene expression profiles in cells from rhesus (Macaca mulatta), cynomolgus (Macaca fascicularis), and pig-tailed (Macaca nemestrina) macaques

• Assess IFNG receptor binding affinity and downstream signaling kinetics across species

• Evaluate differential sensitivity to pathogens following IFNG priming of macrophages from different macaque species

• Measure species-specific variations in antimicrobial responses (e.g., autophagy, phagosome maturation, or reactive oxygen species production)

• Analyze interspecies differences in IFNG-regulated genes that may correlate with disease susceptibility

These comparative approaches can identify species-specific immune mechanisms that may explain differential susceptibility to infectious diseases, particularly relevant for SIV/HIV research .

What factors should be considered when designing dose-response experiments with recombinant rhesus macaque IFNG?

Designing rigorous dose-response experiments with recombinant rhesus macaque IFNG requires consideration of multiple variables:

• Concentration range: Begin with a broad range (e.g., 0.1-1000 ng/mL) based on the expected ED50 of 50-500 ng/mL for most biological effects

• Time-course analysis: Include multiple timepoints (2h, 6h, 24h, 48h) to capture both early and late response genes

• Cell type specificity: Different cell types (lymphocytes, macrophages, epithelial cells) may have varying sensitivities to IFNG

• Readout selection: Choose appropriate readouts based on experimental goals (phospho-STAT1, MHC upregulation, gene expression)

• Synergistic factors: Consider co-stimulation with TNF-α or other cytokines that may potentiate IFNG effects

• Receptor saturation: Include concentrations that ensure receptor saturation to determine maximum response

• Vehicle controls: Include proper controls for the protein buffer components

A robust experimental design would incorporate at least 5-6 concentrations in logarithmic scale with minimum 3 biological replicates per condition.

How should researchers validate the specificity of recombinant rhesus macaque IFNG-induced effects?

Validating the specificity of observed effects requires multiple experimental approaches:

• Receptor blocking: Use neutralizing antibodies against IFNGR1 to demonstrate receptor specificity

• JAK inhibitors: Apply specific JAK1/2 inhibitors (e.g., ruxolitinib) to confirm canonical signaling pathway dependence

• Heat-inactivated controls: Compare responses with heat-denatured IFNG protein

• Competitive inhibition: Use excess unlabeled IFNG to compete with labeled IFNG in binding assays

• Gene knockout validation: When possible, use IFNGR1-knockout cells to confirm receptor dependence

• Species cross-reactivity tests: Compare with human IFNG to identify species-specific versus conserved responses

• Pathway-specific gene expression: Analyze established IFNG-responsive genes like CXCL9, CXCL10, IDO1, and GBP1

These validation approaches help distinguish IFNG-specific effects from non-specific protein or contaminant-induced responses, ensuring experimental rigor.

What experimental considerations are important when using recombinant IFNG in mucosal tissue explant cultures?

Working with mucosal tissue explants presents unique challenges for IFNG stimulation experiments:

• Tissue viability: Validate explant viability before and after IFNG treatment using LDH release assays or metabolic indicators

• Penetration kinetics: Consider the time required for IFNG to penetrate tissue architecture (generally longer than cell monolayers)

• Polarized application: For epithelial tissues, apply IFNG to either apical or basolateral surfaces based on research question

• Endogenous IFNG baseline: Measure baseline IFNG expression, as mucosal tissues constitutively express certain interferons

• Co-stimulatory factors: Include relevant mucosal cytokines (IL-22, IL-17) that may interact with IFNG signaling

• 3D culture systems: Consider organoid cultures for improved physiological relevance

• Barrier integrity assessment: Monitor epithelial barrier function using TEER measurements during IFNG treatment

These considerations ensure that experiments with mucosal tissues accurately recapitulate in vivo conditions and produce interpretable results.

How can phosphoproteomics be used to compare rhesus macaque versus human IFNG signaling cascades?

Phosphoproteomic analysis offers powerful insights into species-specific differences in IFNG signaling:

• Sample preparation methodology:

  • Stimulate matched cell types (e.g., macrophages) from both species with IFNG

  • Harvest cells at multiple early timepoints (5, 15, 30, 60 minutes)

  • Extract phosphopeptides using TiO2 or IMAC enrichment

  • Perform LC-MS/MS analysis with isobaric labeling (TMT or iTRAQ)

• Data analysis approach:

  • Identify differentially phosphorylated proteins between species

  • Map phosphorylation sites to conserved signaling networks

  • Perform kinase activity prediction based on substrate phosphorylation

  • Validate key differences using phospho-specific antibodies

• Key pathways to examine:

  • JAK-STAT canonical pathway components

  • Non-canonical pathways (MAPK, PI3K, NF-κB)

  • Negative regulatory pathways (SOCS, PIAS)

  • Species-specific signaling nodes

This approach can reveal subtle mechanistic differences that explain species-specific immune responses and improve translation of macaque studies to human applications.

What are the challenges in comparing transcriptional responses to IFNG between rhesus macaque and human cells?

Comparative transcriptomics between species presents several methodological challenges:

• Reference genome considerations:

  • Ensure comparable genome annotation quality between species

  • Account for gene duplication events unique to each species

  • Consider alternative splicing differences that affect transcript quantification

• Homology mapping issues:

  • Some genes lack clear one-to-one orthologs

  • Paralogs may have diverged in function between species

  • Non-coding RNAs often show poor conservation

• Technical standardization:

  • Cell type equivalence must be ensured

  • Identical stimulation conditions required

  • Batch effects must be controlled between species

• Data analysis approaches:

  • Use ortholog-based gene set enrichment methods

  • Employ network-based comparisons rather than individual gene comparisons

  • Focus on pathway-level responses rather than individual genes

  • Validate key differences with species-specific qPCR

Researchers should emphasize conserved pathway responses while acknowledging that individual gene components may differ between species.

How can single-cell approaches enhance understanding of rhesus macaque IFNG responses in heterogeneous immune populations?

Single-cell technologies provide unprecedented resolution of IFNG responses across immune cell subsets:

• Experimental design considerations:

  • IFNG stimulation of PBMCs or tissue-derived cell suspensions

  • Time-series analysis to capture response kinetics

  • Integration with cell surface phenotyping (CITE-seq)

  • Paired TCR/BCR sequencing when relevant

• Technical approaches:

  • scRNA-seq to capture transcriptional heterogeneity

  • scATAC-seq to identify chromatin accessibility changes

  • Phospho-protein detection at single-cell level (CyTOF)

  • Spatial transcriptomics to preserve tissue context

• Data analysis strategies:

  • Trajectory analysis to map response evolution

  • Identification of responder vs. non-responder populations

  • Regulatory network reconstruction

  • Integration with bulk phosphoproteomics/proteomics

• Biological insights gained:

  • Cell type-specific IFNG response thresholds

  • Identification of previously unknown responsive cell populations

  • Characterization of resistance mechanisms in non-responsive cells

  • Temporal ordering of IFNG-induced gene programs

These approaches overcome limitations of bulk analyses that mask cell type-specific responses and reveal the true complexity of IFNG biology in primate immune systems.

What are common pitfalls when measuring IFNG-induced biological effects and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant IFNG:

What reconstitution and handling procedures ensure optimal recombinant rhesus macaque IFNG activity?

Proper handling of recombinant proteins is critical for maintaining biological activity:

• Reconstitution protocol:

  • For carrier-containing IFNG: Reconstitute at 25 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

  • For carrier-free IFNG: Reconstitute at 100 μg/mL in sterile PBS

  • Allow protein to dissolve completely (15-20 minutes at room temperature)

  • Mix by gentle swirling rather than vortexing to avoid denaturation

• Handling practices:

  • Use low-protein binding tubes and pipette tips

  • Prepare working dilutions immediately before use

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Store reconstituted protein at -20°C for short-term or -80°C for long-term storage

• Working dilution preparation:

  • Dilute in appropriate serum-free or complete medium depending on experiment

  • For prolonged incubations, consider medium supplementation with stabilizers

  • Filter-sterilize if necessary using low-protein binding 0.22 μm filters

These procedures maximize protein stability and experimental reproducibility when working with recombinant rhesus macaque IFNG .

How should researchers normalize and compare IFNG potency between different experimental systems?

Standardizing IFNG potency measurements across experimental systems requires rigorous analytical approaches:

• Activity standardization methods:

  • Define specific activity units based on a standard bioassay (e.g., induction of MHC II on macrophages)

  • Calculate EC50 values for each lot in a reference assay system

  • Express potency relative to an international standard where available

• Cross-experiment normalization strategies:

  • Include internal reference controls in each experiment

  • Calculate fold-change relative to untreated controls rather than absolute values

  • Use area-under-curve (AUC) analysis for dose-response rather than single points

  • Normalize to maximal response when receptor numbers vary between systems

• Statistical considerations:

  • Apply four-parameter logistic regression for dose-response analysis

  • Use relative potency calculations rather than direct EC50 comparisons

  • Account for system-specific factors (receptor density, negative regulator levels)

These approaches enable meaningful comparisons of IFNG activity across different experimental systems, cell types, and studies.

What approaches can address contradictory findings between in vitro IFNG studies and in vivo observations in rhesus macaques?

Reconciling in vitro and in vivo discrepancies requires systematic investigation:

• Mechanistic reconciliation approaches:

  • Examine concentration differences (in vitro doses often exceed physiological levels)

  • Consider timing differences (acute vs. chronic exposure)

  • Assess compensatory mechanisms present in vivo but absent in vitro

  • Evaluate contributions of the tissue microenvironment and cell-cell interactions

• Experimental strategies:

  • Employ ex vivo assays using freshly isolated cells/tissues

  • Develop complex 3D culture systems that better mimic in vivo conditions

  • Perform parallel in vitro and in vivo studies with matched readouts

  • Consider organotypic cultures that preserve tissue architecture

• Analytical framework:

  • Develop integrated models that account for pharmacokinetics/pharmacodynamics

  • Use systems biology approaches to identify network-level differences

  • Compare IFNG signatures rather than individual gene responses

  • Validate in vitro findings with targeted in vivo experiments

This systematic approach helps bridge the gap between reductionist in vitro systems and complex in vivo biology.