Recombinant Rhesus Macaque CSF3 protein (CSF3), partial (Active)

What is Recombinant Rhesus Macaque CSF3 protein and how does it relate to human CSF3?

Recombinant Rhesus Macaque CSF3 protein is a biologically active cytokine produced in E. coli expression systems that corresponds to amino acids 27-200 of the native rhesus macaque CSF3 sequence (UniProt: F7H1Q6). It is the macaque homolog of human Granulocyte Colony-Stimulating Factor (G-CSF). The recombinant protein is tag-free with a molecular weight of approximately 18.9 kDa . While human and rhesus CSF3 share significant sequence homology, species-specific differences exist that make rhesus CSF3 particularly valuable for preclinical studies using rhesus macaque models. The protein functions primarily to stimulate the production, differentiation, and functional activation of neutrophils, similar to its human counterpart.

What are the structural features of rhesus macaque CSF3 and how do they influence function?

The recombinant rhesus macaque CSF3 spans amino acids 27-200 of the native sequence with the following primary structure: "TPLGPASSLP QSFLLKCLEQ VRKIQGDGAA LQEKLCATYK LCHPEELVLL RHSLGIPWAP LSSCPSQALQ LTGCLSQLHS SLFLYQGLLQ ALEGISPELS PTLDTLQLDI ADFATTIWQQ MEDLGMAPAL QPTQGAMPAF TSAFQRRAGG VLVASHLQRF LELAYRVLRH LAQS" . This structure contains four alpha-helices in an up-up-down-down topology with two intrachain disulfide bonds that are crucial for maintaining the proper three-dimensional conformation and biological activity. The N-terminal region (amino acids 27-40) contains elements critical for receptor binding, while the C-terminal region contributes to protein stability. Unlike some recombinant proteins, this particular preparation is non-glycosylated as it is produced in E. coli, which may affect certain pharmacokinetic properties compared to the native glycosylated form, though the core biological activity remains intact.

How is the biological activity of recombinant rhesus macaque CSF3 measured and what are the typical activity values?

The biological activity of recombinant rhesus macaque CSF3 is typically quantified using cell proliferation assays with factor-dependent cell lines. Specifically, the ED50 (effective dose for 50% maximal response) is determined using murine NFS-60 cells, which depend on CSF3 for proliferation. According to specification data, the recombinant rhesus macaque CSF3 exhibits an ED50 of less than 0.05 ng/mL, corresponding to a specific activity of approximately 2.0 × 10^7 IU/mg . This high specific activity indicates the protein's potent biological effects at low concentrations.

The methodology involves:

• Culturing NFS-60 cells in the presence of serial dilutions of the recombinant protein

• Measuring cell proliferation after 48-72 hours using colorimetric assays (e.g., MTT or WST-1)

• Calculating the ED50 by plotting the dose-response curve

• Comparing activity to a reference standard to determine specific activity in International Units (IU)

This standardized biological activity measurement ensures consistency between different production lots and serves as a quality control parameter for research applications.

How can recombinant rhesus macaque CSF3 be used to study immune responses in rhesus macaque models?

Recombinant rhesus macaque CSF3 provides a valuable tool for studying neutrophil-mediated immune responses in rhesus macaque models. Unlike human CSF3 used in macaque studies, the species-matched recombinant protein avoids potential immunogenicity issues that could confound results. Researchers can administer the protein subcutaneously or intravenously to observe effects on neutrophil production, mobilization, and function in the context of infectious disease models, vaccine studies, or inflammatory conditions.

Based on comparable studies with colony stimulating factors in rhesus macaques, administration typically leads to a 2-3 fold increase in peripheral white blood cells within 24 hours, with neutrophils contributing to 50-80% of this increase . The biological effects are dose-dependent and persist throughout the treatment period. Upon discontinuation, white blood cell counts typically return to baseline within one week . This predictable kinetic profile makes it useful for precisely timed experimental manipulations of the immune system.

Additionally, CSF3 can enhance the functional activity of mature neutrophils, including oxidative metabolism and bactericidal activity , providing researchers with the ability to study not just numerical but also qualitative changes in neutrophil populations during immune responses.

What experimental designs best evaluate CSF3 effects in viral infection models using rhesus macaques?

When evaluating CSF3 effects in viral infection models using rhesus macaques, researchers should consider multi-armed study designs that isolate the specific contribution of CSF3 to antiviral immunity. Based on studies examining colony stimulating factor biology in macaques, the following experimental design elements are recommended:

• Treatment timing: Administer recombinant rhesus macaque CSF3 either prophylactically (before viral challenge) or therapeutically (after established infection) with appropriate control groups.

• Dosing regimen: Consider subcutaneous administration (three times daily), which has shown superior efficacy compared to intravenous infusion for colony stimulating factors . Typical dosing ranges from 5-20 μg/kg based on comparable studies.

• Sampling schedule: Collect serial blood samples for:

  • Complete blood counts to monitor neutrophil kinetics

  • Flow cytometry to assess neutrophil activation markers

  • Functional assays (oxidative burst, phagocytosis, NET formation)

  • Viral load measurements to correlate with neutrophil parameters

• Tissue analysis: Include tissue sampling (lymph nodes, mucosal tissues) to evaluate neutrophil infiltration and interaction with virus-infected cells.

• Virological endpoints: Measure viral loads in blood and tissues to assess the impact of CSF3 treatment on viral containment or clearance.

The particular value of the rhesus macaque model is demonstrated in studies showing that viral proteins (like BARF1 from lymphocryptoviruses) can block colony stimulating factor signaling, affecting viral loads during both acute and persistent infection phases . This suggests that CSF3-mediated immune responses may be actively targeted by viral immune evasion mechanisms, making it an important pathway to investigate in viral pathogenesis.

How can recombinant rhesus macaque CSF3 be incorporated into vaccine immunogenicity studies?

Recombinant rhesus macaque CSF3 can serve as both an immunological adjuvant and an investigational tool in vaccine studies. Strategic implementation includes:

• As an adjuvant: Co-administration of CSF3 with vaccine antigens can enhance neutrophil-mediated innate immune responses that subsequently shape adaptive immunity. Administration should begin 1-2 days before vaccination and continue for 3-5 days post-vaccination to maximize the innate immune response during antigen presentation.

• For mechanistic studies: Administering CSF3 during specific windows relative to vaccination allows researchers to dissect the role of neutrophils in vaccine-induced immunity:

  • Early phase (0-3 days post-vaccination): Investigate innate immune contributions

  • Late phase (7+ days post-vaccination): Examine effects on adaptive immune development

• Experimental readouts should include:

  • Neutrophil infiltration at vaccination sites

  • Neutrophil interaction with antigen-presenting cells

  • Impact on antigen transport to lymph nodes

  • Effects on germinal center formation

  • Alterations in antibody subclass distribution

The importance of colony stimulating factors in shaping immune responses is highlighted by research showing that viral interruption of CSF-1 signaling significantly impacts both acute infection dynamics and persistent viral setpoints . This suggests CSF3 may similarly influence vaccine-induced immunity through modulation of innate immune responses.

When analyzing antibody responses in these studies, researchers should consider that rhesus macaque antibody effector functions may not directly parallel their human counterparts. For example, unlike in humans where IgG1 and IgG3 dominate effector functions, in rhesus macaques, IgG1 exhibits the greatest effector function activity, followed by IgG2 and then IgG3/4 .

What are the optimal reconstitution and storage conditions for recombinant rhesus macaque CSF3?

The optimal handling of recombinant rhesus macaque CSF3 is critical for maintaining its biological activity. The protein is typically supplied as a lyophilized powder that requires careful reconstitution and storage as follows:

• Reconstitution procedure:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (recommended: 50%) for long-term storage

  • Mix gently by swirling rather than vortexing to prevent protein denaturation

• Storage conditions:

  • Long-term storage: Aliquot and store at -20°C to -80°C

  • Working stocks: Store at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles as this significantly reduces biological activity

• Quality verification after reconstitution:

  • Visual inspection for clarity (solution should be clear without particulates)

  • Verify protein concentration by spectrophotometric methods

  • Consider running a small-scale bioactivity assay when using for critical experiments

The reconstituted protein has been verified to maintain >98% purity as determined by SDS-PAGE and HPLC analysis, with endotoxin levels less than 1.0 EU/μg . These specifications ensure that experimental outcomes are not influenced by contaminants or loss of biological activity.

What dose-response relationships and pharmacokinetics should be considered when designing in vivo rhesus macaque experiments?

When designing in vivo experiments with recombinant rhesus macaque CSF3 in macaque models, researchers should consider the following dose-response relationships and pharmacokinetic factors:

• Dose-response considerations:

  • Initial response threshold: Based on studies with similar colony stimulating factors, effects on peripheral blood cell counts begin to appear at doses as low as 1-3 μg/kg

  • Dose-dependent response: White blood cell increases occur in a dose-dependent manner, with maximum effects typically observed at 10-15 μg/kg

  • Saturation effects: Doses exceeding 20 μg/kg may not provide proportionally greater effects due to receptor saturation

• Pharmacokinetic parameters:

  • Route-dependent bioavailability: Subcutaneous administration typically results in more effective leukocytosis compared to intravenous administration

  • Half-life: Non-glycosylated recombinant CSF3 (E. coli-derived) has a shorter circulatory half-life (2-4 hours) compared to glycosylated forms

  • Dosing frequency: Multiple daily doses (e.g., three times daily for subcutaneous administration) maintain more stable blood levels and produce more consistent effects

• Temporal response pattern:

  • Initial response: 2-3 fold increase in white blood cells within 24 hours

  • Peak response: Maximum levels typically reached on the last day of treatment or one day after

  • Return to baseline: Cell counts normalize within approximately one week after treatment cessation

This data should be used to design treatment protocols with appropriate dosing intervals and duration to achieve the desired biological effect for the specific research question being addressed.

How does the E. coli-expressed non-glycosylated form of CSF3 compare to glycosylated versions for experimental applications?

The E. coli-expressed recombinant rhesus macaque CSF3 is non-glycosylated due to the prokaryotic expression system's inability to perform post-translational glycosylation. This creates important distinctions from glycosylated forms that researchers should consider:

Research comparing glycosylated and non-glycosylated recombinant human GM-CSF in rhesus monkeys demonstrated that both forms effectively stimulated leukocytosis and enhanced functional activity of granulocytes in vivo . This suggests that non-glycosylated CSF3 is suitable for most experimental applications, particularly when administered subcutaneously and with appropriate dosing frequency to compensate for the shorter half-life.

For experiments requiring sustained CSF3 action over extended periods, researchers may need to implement more frequent dosing schedules with the non-glycosylated form or consider alternative glycosylated versions produced in mammalian expression systems.

How can recombinant rhesus macaque CSF3 be used to investigate viral immune evasion mechanisms?

Recombinant rhesus macaque CSF3 provides a sophisticated tool for investigating viral immune evasion strategies targeting myeloid cell function. Research has demonstrated that viruses like Epstein-Barr virus (and its rhesus macaque counterpart, rhLCV) encode proteins that specifically interfere with colony stimulating factor signaling pathways to establish successful infection .

Experimental approaches to investigate viral immune evasion include:

• Competitive binding studies: Determine whether viral proteins directly interact with CSF3 or its receptor using:

  • Surface plasmon resonance to measure binding kinetics

  • Co-immunoprecipitation assays from infected cells

  • Fluorescently labeled CSF3 to visualize receptor binding in the presence/absence of viral proteins

• Signaling pathway analysis: Examine whether viral proteins disrupt CSF3-induced signaling cascades by measuring:

  • JAK/STAT phosphorylation events

  • Downstream transcriptional responses using RNA-seq

  • Protein expression changes in neutrophil effector molecules

• Functional neutrophil assays: Assess whether viral interference with CSF3 impairs neutrophil functions:

  • Oxidative burst capacity

  • Phagocytosis efficiency

  • Neutrophil extracellular trap (NET) formation

  • Bactericidal activity

• In vivo challenge models: Use recombinant viruses with mutations in CSF-targeting viral genes alongside CSF3 administration to determine:

  • Whether CSF3 supplementation can overcome viral immune evasion

  • If timing of CSF3 administration relative to infection alters viral clearance

Studies with rhLCV demonstrated that viral BARF1-mediated blockade of CSF-1 significantly impacted both acute viral loads and the establishment of viral setpoints during persistent infection . Similar mechanisms may exist for CSF3, and elucidating these could provide new targets for antiviral strategies. The ability to study these interactions in rhesus macaques with species-matched CSF3 offers unique translational insights not possible in other model systems.

What are the methodological approaches for comparing CSF3 signaling between human and rhesus macaque systems?

Comparing CSF3 signaling between human and rhesus macaque systems requires sophisticated comparative methodologies that can identify both conserved and divergent aspects of this critical cytokine pathway. The following approaches are recommended:

• Receptor binding kinetics comparison:

  • Express recombinant human and rhesus CSF3 receptors

  • Measure binding affinity (KD), association (kon) and dissociation (koff) rates using surface plasmon resonance

  • Cross-species binding experiments (human CSF3 to rhesus receptor and vice versa)

  • Competition binding assays to determine receptor specificity

• Signaling pathway analysis:

  • Phosphoproteomic profiling following CSF3 stimulation in human vs. rhesus cells

  • Time-course analysis of JAK/STAT activation

  • RNA-seq to compare transcriptional responses across species

  • CRISPR-based genetic screens to identify species-specific pathway components

• Functional cross-comparison:

  • Side-by-side neutrophil functional assays using both species' cells

  • Dose-response curves for multiple neutrophil functions

  • Inhibitor sensitivity profiling to identify differential pathway dependencies

  • Ex vivo migration and chemotaxis assays

• Structural biology approaches:

  • Comparative structural analysis of human vs. rhesus CSF3 and receptors

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Molecular dynamics simulations to predict species-specific interaction differences

These comparative approaches are particularly important because studies with rhesus macaque IgG have revealed that despite high sequence homology with human counterparts, functional differences exist in effector activities . Similar nuanced differences may exist in CSF3 biology that could impact the translational relevance of rhesus macaque studies to human applications.

How can recombinant rhesus macaque CSF3 be engineered for enhanced experimental applications?

Advanced protein engineering techniques can modify recombinant rhesus macaque CSF3 to create enhanced variants for specialized experimental applications:

• Half-life extension strategies:

  • PEGylation: Conjugation of polyethylene glycol molecules to specific residues

  • Fc-fusion: Genetic fusion with rhesus IgG Fc domain

  • HSA-fusion: Genetic fusion with human serum albumin

  • Glycoengineering: Introduction of additional glycosylation sites

• Receptor specificity modifications:

  • Site-directed mutagenesis of key receptor-binding residues

  • Domain swapping with human CSF3 to create chimeric proteins

  • Affinity maturation through directed evolution approaches

• Functional enhancements:

  • Engineering variants with enhanced stability at physiological temperature

  • Creating antagonist versions to block endogenous CSF3 signaling

  • Developing bispecific molecules that target CSF3 activity to specific tissues

• Trackable variants for in vivo imaging:

  • Genetic fusion with fluorescent proteins that maintain biological activity

  • Introduction of bioorthogonal chemistry handles for subsequent labeling

  • Dual-function variants that both activate the receptor and enable tracking

When engineering such variants, researchers should conduct comprehensive functional validation including:

• Receptor binding assays compared to wild-type protein

• Cell-based proliferation assays using NFS-60 cells

• Assessment of pharmacokinetic properties in preliminary animal studies

• Verification of neutrophil activation profiles

The engineering approach should be tailored to the specific experimental question. For instance, studying CSF3's role during viral infection might benefit from longer-acting variants, while mechanistic studies exploring receptor dynamics might require fluorescently tagged versions with minimal perturbation to binding kinetics.

What are common technical challenges when working with recombinant rhesus macaque CSF3 and how can they be addressed?

Researchers working with recombinant rhesus macaque CSF3 may encounter several technical challenges that can impact experimental outcomes. Here are common issues and their solutions:

• Loss of biological activity during storage/handling:

  • Problem: Protein aggregation or denaturation

  • Solution: Add carrier protein (0.1-0.5% BSA) to dilute solutions, avoid vigorous agitation, prepare fresh working stocks weekly, store concentrated aliquots with 50% glycerol at -80°C

• Inconsistent cellular responses:

  • Problem: Variable receptor expression on target cells

  • Solution: Pre-screen cell populations for CSF3 receptor expression, standardize cell culture conditions, use internal control conditions in each experiment

• Lot-to-lot variability:

  • Problem: Different specific activities between production lots

  • Solution: Calibrate each new lot against a reference standard using a bioactivity assay, normalize dosing based on specific activity rather than protein mass

• Endotoxin contamination effects:

  • Problem: Bacterial endotoxin co-purification affecting immune cell responses

  • Solution: Verify endotoxin levels (<1.0 EU/μg) , include polymyxin B controls in sensitive assays, use endotoxin removal columns if necessary

• Protein adsorption to labware:

  • Problem: Loss of protein due to binding to plastic surfaces

  • Solution: Use low-binding tubes and pipette tips, pre-coat surfaces with dilute protein solution, include carrier protein in working solutions

• Suboptimal reconstitution:

  • Problem: Incomplete solubilization of lyophilized protein

  • Solution: Follow precise reconstitution protocol, centrifuge vial before opening, allow adequate time for complete dissolution, filter solution if necessary

Implementing these solutions will help ensure consistent and reliable results when working with recombinant rhesus macaque CSF3 in various experimental applications.

How should researchers design appropriate controls when using recombinant rhesus macaque CSF3 in complex experimental systems?

Robust experimental design with appropriate controls is essential when using recombinant rhesus macaque CSF3 in complex systems. The following control strategies should be implemented:

• Vehicle controls:

  • Buffer-only control containing all components used in CSF3 reconstitution

  • Match glycerol concentration and carrier protein if used

  • Process control samples identically through all experimental steps

• Biological activity controls:

  • Heat-inactivated CSF3 (95°C for 10 minutes) to control for non-specific protein effects

  • Concentration-matched irrelevant protein control (e.g., recombinant GFP)

  • Validated human CSF3 as a positive control for comparative studies

• Receptor specificity controls:

  • CSF3 receptor blocking antibodies to confirm signaling specificity

  • CSF3 neutralizing antibodies added to protein preparations

  • Cells lacking CSF3 receptor expression as negative biological controls

• In vivo experimental controls:

  • Within-subject controls where feasible (e.g., contralateral administration)

  • Time-matched sampling from pre-treatment baseline

  • Separate vehicle control cohorts matched for age, sex, and weight

  • Staggered treatment schedules to control for environmental variables

• Technical validation controls:

  • Spike-in recovery controls for complex biological fluids

  • Standard curves encompassing expected concentration ranges

  • Inter-assay calibrators for studies requiring multiple analytical runs

Research with rhLCV demonstrated the importance of proper controls by comparing wild-type virus to recombinant virus with a mutated BARF1 gene, and then to a repaired recombinant virus, conclusively demonstrating the specific effect of BARF1-mediated CSF-1 blockade on viral loads . Similarly rigorous control strategies should be employed when studying CSF3 biology.

What analytical methods are most appropriate for measuring CSF3-induced neutrophil responses in rhesus macaque models?

Comprehensive assessment of CSF3-induced neutrophil responses in rhesus macaque models requires multi-parameter analytical approaches that capture both quantitative and qualitative aspects of neutrophil biology:

• Quantitative measurements:

  • Complete blood counts with differential to track neutrophil numbers

  • Flow cytometry with rhesus-specific antibodies for neutrophil subpopulation analysis

  • Tissue neutrophil quantification by immunohistochemistry with anti-neutrophil markers

  • Absolute neutrophil count tracking using reference beads in flow cytometry

• Functional assays:

  • Oxidative burst measurement using dihydrorhodamine 123 or luminol-enhanced chemiluminescence

  • Phagocytosis assays using fluorescent bacterial particles

  • Bacterial killing assays with clinically relevant pathogens

  • Neutrophil extracellular trap (NET) quantification by fluorescence microscopy or ELISA

• Molecular analyses:

  • Transcriptomic profiling of isolated neutrophils using RNA-seq

  • Neutrophil proteome analysis focusing on granule components

  • Phosphoprotein signaling cascade measurement using phospho-flow cytometry

  • Epigenetic analysis of neutrophil population shifts

• Imaging approaches:

  • Intravital microscopy of neutrophil trafficking in accessible tissues

  • PET imaging with radiolabeled markers of neutrophil activity

  • Two-photon microscopy of ex vivo tissue samples

  • Whole-body distribution studies using labeled neutrophils

Studies with colony stimulating factors in rhesus macaques have shown that beyond increasing white blood cell counts by 2-3 fold, these factors enhance neutrophil oxidative metabolism and bactericidal activity . Therefore, comprehensive analysis should include both quantitative metrics (cell numbers) and functional parameters (killing capacity, respiratory burst) to fully characterize CSF3-induced responses.

When conducting these analyses, it is essential to establish baseline values for individual animals before treatment, as substantial inter-individual variation in neutrophil parameters exists in rhesus macaques.