Recombinant Macaca mulatta Interleukin-1 alpha (IL1A) (Active)

What is the biological function of Macaca mulatta Interleukin-1 alpha in immune responses?

Macaca mulatta Interleukin-1 alpha (IL1A) functions as a potent pro-inflammatory cytokine primarily produced by activated macrophages. Its primary biological activities include stimulation of thymocyte proliferation through the induction of IL-2 release, promotion of B-cell maturation and proliferation, and enhancement of fibroblast growth factor activity. IL1A plays a central role in inflammatory responses as an endogenous pyrogen, mediating fever responses and other systemic inflammatory effects. Additionally, IL1A has been documented to stimulate the release of prostaglandins and collagenase from synovial cells, contributing to inflammatory pathologies involving joint tissues .

How does the amino acid sequence of rhesus macaque IL1A compare to human IL1A?

The amino acid sequence of rhesus macaque IL1A exhibits significant homology with human IL1A, though with distinct species-specific differences. The full-length protein has an expression region spanning amino acids 113-271 with the sequence: SAPFSFLSNMTYHFIRIIKHEFILNDTLNQTIIRANDQHLTAAAIHNLDEAVKFDMGAYTSSKDDTKVPVILRISKTQLYVSAQDEDQPVLLKEMPEINKTITGSETNFLFFWETHGTKNYFISVAHPNLFIATKHDNWVCLAKGLPSITDFQILENQA . Comparative sequence analyses of cytokine genes from human and nonhuman primates, as referenced in Villinger et al. (1995), demonstrate important structural conservation while also highlighting species-specific variations that may influence function and immunological cross-reactivity . These sequence differences are crucial considerations when designing experiments that aim to translate findings between macaque models and human systems.

What are the distinguishing features of active recombinant IL1A compared to the native protein?

Active recombinant Macaca mulatta IL1A retains the functional domains of the native protein while incorporating modifications that facilitate purification and potentially enhance stability. The recombinant protein typically includes an N-terminal tag, such as the 6xHis-SUMO tag in E. coli-derived preparations or a 6xHis tag in yeast-derived versions . These tags enable efficient purification through affinity chromatography while minimizing interference with the protein's biological activity. The molecular weight of the recombinant protein varies based on the expression system used – approximately 34.1 kDa for the E. coli-derived protein with a 6xHis-SUMO tag, compared to approximately 20.1 kDa for the yeast-derived version with a 6xHis tag . While the expression region (amino acids 113-271) corresponds to the mature, biologically active portion of the protein, researchers should validate that the recombinant protein's activity profile matches that of native IL1A through appropriate functional assays.

What expression systems are used for producing recombinant Macaca mulatta IL1A and how do they differ?

Two primary expression systems are commonly employed for producing recombinant Macaca mulatta IL1A: bacterial (E. coli) and yeast-based systems. Each system offers distinct advantages and limitations that researchers should consider when selecting a recombinant protein for their specific experimental requirements.

E. coli-based expression systems typically yield IL1A with an N-terminal 6xHis-SUMO tag, resulting in a protein with a molecular weight of approximately 34.1 kDa . This system offers high protein yields and cost-effective production but may have limitations regarding post-translational modifications. The bacterial expression system is particularly suitable for structural studies and applications where glycosylation patterns are not critical.

In contrast, yeast-based expression systems produce IL1A with an N-terminal 6xHis tag and a molecular weight of approximately 20.1 kDa . Yeast expression provides advantages in terms of eukaryotic post-translational modifications, potentially resulting in a protein conformation that more closely resembles the native mammalian cytokine. This system may be preferable for applications where protein folding and secondary structure are critical considerations.

What methods are used to verify the purity and identity of recombinant Macaca mulatta IL1A?

Multiple complementary analytical techniques are employed to verify the purity and identity of recombinant Macaca mulatta IL1A preparations:

• SDS-PAGE Analysis: This technique is the primary method for assessing protein purity, with commercial preparations typically exceeding 90% purity as determined by this method . The protein band should correspond to the expected molecular weight (34.1 kDa for E. coli-derived or 20.1 kDa for yeast-derived preparations).

• Western Blotting: Immunological verification using specific antibodies against either IL1A or the affinity tag helps confirm protein identity.

• Mass Spectrometry: For definitive confirmation of protein sequence and identification of any post-translational modifications.

• Biological Activity Assays: Functional tests to confirm that the recombinant protein exhibits expected biological activities, such as stimulation of target cells that express IL1A receptors.

• Endotoxin Testing: Particularly important for preparations intended for in vivo applications or primary cell culture experiments, as bacterial endotoxin contamination can confound experimental results when studying inflammatory responses.

How does the molecular weight of recombinant IL1A differ based on the expression system used?

The molecular weight of recombinant Macaca mulatta IL1A varies significantly depending on the expression system employed, primarily due to differences in the fusion tags and post-translational modifications. E. coli-derived recombinant IL1A typically incorporates an N-terminal 6xHis-SUMO tag, resulting in a final molecular weight of approximately 34.1 kDa . The SUMO tag serves to enhance protein solubility and expression efficiency in bacterial systems while facilitating purification.

In contrast, yeast-derived recombinant IL1A utilizes a smaller N-terminal 6xHis tag, yielding a protein with a molecular weight of approximately 20.1 kDa . This difference of approximately 14 kDa between the two preparations is primarily attributable to the size difference between the SUMO and simple His tags. Additionally, yeast expression systems may introduce eukaryotic post-translational modifications not present in bacterial preparations.

These molecular weight differences are important considerations when designing experiments, particularly for applications where protein size may influence diffusion, receptor binding kinetics, or systemic distribution in in vivo models.

How can researchers verify the biological activity of recombinant Macaca mulatta IL1A in vitro?

Researchers can verify the biological activity of recombinant Macaca mulatta IL1A through several complementary functional assays:

• Proliferation Assays: Similar to methods used for other rhesus cytokines like IL-9 and IL-33, researchers can assess the ability of IL1A to enhance the proliferation of responsive cell lines, particularly macaque B cell lines . This typically involves treating cells with various concentrations of the recombinant protein and measuring proliferation via methods such as MTT/XTT assays or thymidine incorporation.

• Cytokine Induction: Measuring the production of secondary cytokines (such as IL-6 or IL-8) from responsive cells following IL1A stimulation using ELISA or multiplex cytokine assays.

• Signaling Pathway Activation: Assessing the phosphorylation status of downstream signaling molecules (such as NF-κB, p38 MAPK, or JNK) using Western blotting or flow cytometry-based phospho-protein detection.

• Gene Expression Analysis: Quantifying the induction of IL1A-responsive genes using RT-PCR or RNA sequencing, similar to approaches used in macaque gene expression studies .

• Receptor Binding Assays: Confirming specific binding to IL-1 receptors using competitive binding assays with labeled ligands or surface plasmon resonance techniques.

A dose-response relationship should be established for each of these assays to determine the EC50 (half-maximal effective concentration) of the recombinant protein, which can serve as a benchmark for batch-to-batch consistency.

What cell types and experimental models are most suitable for studying Macaca mulatta IL1A functions?

Several cell types and experimental models are particularly well-suited for investigating the functions of Macaca mulatta IL1A:

• Primary Macaque Macrophages: As the principal producers of IL1A, macrophages derived from peripheral blood monocytes or tissue-resident populations provide a physiologically relevant system for studying both the production and response to IL1A.

• Macaque Peripheral Blood Mononuclear Cells (PBMCs): These heterogeneous cell populations, including lymphocytes and monocytes, offer a comprehensive model for examining IL1A's effects on multiple immune cell types simultaneously .

• Macaque CD4+ T Cells: While primarily used for studying other cytokines like IL-9, these cells can also respond to IL1A, making them useful for investigating IL1A's role in adaptive immune responses .

• Rhesus B Cell Lines: These can be used to study the proliferative effects of IL1A, similar to approaches used with other rhesus cytokines .

• In vivo Macaca mulatta Models: Particularly valuable for studying systemic inflammatory responses and disease models, similar to the malaria infection models described in the literature .

When selecting an experimental model, researchers should consider the specific biological question being addressed, the availability of reagents specific for macaque systems, and how closely the model recapitulates the human condition if translational relevance is a goal.

What are the optimal concentrations of recombinant IL1A for different experimental applications?

The optimal concentration of recombinant Macaca mulatta IL1A varies significantly depending on the specific experimental application, cell type, and desired biological response. While exact dosing must be empirically determined for each experimental system, the following guidelines can serve as starting points:

Table 1: Recommended Starting Concentrations for Various Applications

For each application, researchers should perform preliminary dose-response experiments covering a concentration range spanning at least 3-4 orders of magnitude to identify both the threshold for biological activity and potential inhibitory effects at high concentrations. The biological activity of different preparations may vary based on the expression system used (E. coli vs. yeast) , necessitating calibration when switching between preparation types.

What are the recommended storage conditions for maintaining the stability of recombinant Macaca mulatta IL1A?

Proper storage is critical for maintaining the stability and biological activity of recombinant Macaca mulatta IL1A. According to product specifications, the shelf life of recombinant IL1A is influenced by multiple factors including storage state, buffer composition, storage temperature, and the inherent stability of the protein .

The recommended storage conditions are:

• Liquid Formulation: Store at -20°C or preferably -80°C for up to 6 months. The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which helps prevent freeze-thaw damage .

• Lyophilized Formulation: Store at -20°C or -80°C for up to 12 months. Reconstitution should follow manufacturer's specifications, typically using sterile water or a compatible buffer .

• Working Aliquots: For short-term use, store working aliquots at 4°C for no more than one week to minimize activity loss .

Importantly, repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of biological activity . To prevent this, it is strongly recommended to prepare small single-use aliquots upon first thawing of the stock solution.

How should researchers prepare and handle recombinant IL1A to preserve its biological activity?

To maintain the biological activity of recombinant Macaca mulatta IL1A during experimental procedures, researchers should follow these handling guidelines:

• Aliquoting Protocol:

  • Thaw the stock solution on ice slowly and completely

  • Under sterile conditions, prepare small single-use aliquots (typically 5-10 μL)

  • Use low-protein binding tubes to prevent adherence loss

  • Flash-freeze aliquots in liquid nitrogen before transferring to -80°C storage

• Reconstitution of Lyophilized Protein:

  • Use sterile, filtered buffer as recommended by the manufacturer

  • Reconstitute gently by swirling rather than vortexing to avoid protein denaturation

  • Allow complete dissolution before use or aliquoting

• Experimental Handling:

  • Keep the protein on ice during experiment preparation

  • Include carrier protein (typically 0.1-0.5% BSA) in working solutions to prevent non-specific binding to tubes and loss of effective concentration

  • Use within 24 hours of dilution in aqueous buffer without carrier protein

  • Protect from prolonged exposure to light

• Quality Control:

  • Consider including positive controls to verify biological activity in each experimental setup

  • For critical experiments, measure protein concentration after dilution to ensure accuracy

These handling procedures help maintain the structural integrity and functional activity of the recombinant protein, ensuring reliable and reproducible experimental results.

What buffer conditions optimize the stability and activity of recombinant Macaca mulatta IL1A?

The optimal buffer conditions for recombinant Macaca mulatta IL1A stability balance physiological compatibility with protein preservation properties. Commercial preparations typically use a Tris-based buffer containing 50% glycerol for long-term storage . This high glycerol concentration prevents ice crystal formation during freezing and helps maintain protein structure.

For experimental applications, consider the following buffer recommendations:

Table 2: Buffer Conditions for Various Applications

When transitioning from storage to experimental conditions, dilution should be performed carefully to avoid protein denaturation from rapid changes in glycerol concentration or pH. A step-wise dilution approach is recommended for applications requiring high protein concentrations with minimal glycerol content.

Certain conditions should be avoided, including:

• Acidic pH (<6.5)

• High salt concentrations (>300 mM NaCl)

• Chelating agents that may disrupt structural stability

• Detergents that can denature protein structure

How does recombinant Macaca mulatta IL1A compare functionally to human IL1A in experimental systems?

While rhesus macaque IL1A shares substantial homology with human IL1A, important functional differences exist that researchers must consider when designing translational studies. Comparative analysis of cytokine genes from human and nonhuman primates by Villinger et al. (1995) provides foundational insights into these differences .

Functional Similarities:

• Both human and rhesus IL1A function as pro-inflammatory cytokines with similar receptor binding properties

• Both induce thymocyte proliferation and stimulate IL-2 release

• Both promote B-cell maturation and proliferation

• Both function as endogenous pyrogens

Important Differences:

• Receptor Binding Kinetics: Subtle sequence differences may result in altered binding affinity to IL-1 receptors

• Species-Specific Cell Responses: The magnitude and kinetics of cellular responses may differ between human and macaque systems

• Cross-Reactivity: While human IL1A often shows activity in macaque systems, the reverse cross-reactivity may be more limited

• Signaling Pathway Activation: Potential differences in the downstream signaling cascade activation strength and duration

When conducting comparative studies, researchers should ideally include parallel experiments with both human and macaque IL1A to calibrate response differences. This approach is particularly important when using macaque models to predict human responses in drug development or when studying the mechanistic details of IL1A-mediated inflammation.

What experimental designs are most effective for studying IL1A-mediated inflammatory responses in rhesus macaque disease models?

Designing robust experiments to study IL1A-mediated inflammation in rhesus macaque models requires careful consideration of multiple factors:

• Longitudinal Sampling Approach: Similar to the malaria infection study design, collecting samples at multiple timepoints throughout disease progression allows tracking of dynamic IL1A responses . This approach enables identification of both acute and chronic inflammatory phases.

• Integrated Multi-parameter Analysis: Combining measurements of IL1A levels with other inflammatory markers, clinical parameters, and functional readouts provides a comprehensive view of the inflammatory process. This approach was effectively demonstrated in macaque infection studies where gene expression was correlated with parasitemia measurements .

• Intervention Studies: Designing experiments with interventions that modulate IL1A activity (such as IL-1 receptor antagonists or neutralizing antibodies) at different disease stages helps establish causality rather than mere correlation.

• Tissue-Specific Sampling: Collecting samples from multiple tissues rather than only peripheral blood provides insight into tissue-specific IL1A responses, particularly important for localized inflammatory processes.

• Multi-omics Integration: Combining IL1A protein measurements with transcriptomics, as demonstrated in malaria infection studies , and potentially proteomics or metabolomics provides a systems biology perspective on inflammatory cascades.

The most effective experimental designs include appropriate controls, sufficient sample sizes for statistical power, and incorporate both molecular and functional readouts to link IL1A activity with disease pathology.

What are the key considerations when comparing results from different recombinant IL1A preparations in experimental studies?

When comparing results obtained using different recombinant Macaca mulatta IL1A preparations, researchers must account for several variables that could influence experimental outcomes:

• Expression System Differences: E. coli-derived IL1A (34.1 kDa with 6xHis-SUMO tag) versus yeast-derived IL1A (20.1 kDa with 6xHis tag) may exhibit different specific activities due to protein folding differences and post-translational modifications.

• Specific Activity Calibration: Different preparations should be calibrated using standardized bioassays to determine relative specific activity, allowing for dose adjustments based on biological activity rather than protein mass.

• Tag Effects: The presence of different fusion tags (SUMO versus simple His-tag) may affect protein stability, receptor binding, or even introduce steric hindrance in certain experimental systems.

• Endotoxin Contamination: Bacterial-derived preparations may contain varying levels of endotoxin despite purification, which can confound results in inflammation studies. Endotoxin testing and removal is particularly critical.

• Buffer Composition: Differences in storage buffer composition between preparations can affect protein stability and experimental outcomes, especially when the buffer itself is carried into experimental systems.

• Batch-to-Batch Variation: Even within the same expression system and manufacturer, batch-to-batch variations can occur that necessitate calibration between preparations.

To address these variables, researchers should consider:

• Including internal calibration standards when changing preparations

• Running parallel experiments with both preparations during transition periods

• Maintaining detailed records of preparation sources and lot numbers

• Performing batch-specific activity validation before use in critical experiments

How can researchers utilize recombinant Macaca mulatta IL1A in comparative immunology studies?

Recombinant Macaca mulatta IL1A serves as a valuable tool in comparative immunology studies, enabling researchers to explore evolutionary conservation and divergence of immune responses between species. Effective utilization strategies include:

• Cross-Species Receptor Activation Studies: Systematically testing rhesus IL1A on cells from different primate species, including humans, helps map receptor recognition patterns and signaling thresholds across evolutionary distance. This approach reveals both conserved immune pathways and species-specific adaptations.

• Comparative Genomics Integration: Combining functional studies using recombinant IL1A with comparative sequence analysis of IL1A genes and their receptors across primate species, as pioneered by Villinger et al. , provides insight into structure-function relationships.

• Ex vivo Comparative Systems: Developing parallel experimental systems using matched cell types from multiple primate species (including humans and rhesus macaques) allows direct comparison of IL1A responses while controlling for experimental variables.

• Modeling Evolution of Inflammatory Responses: Using recombinant IL1A from multiple species in standardized assays to reconstruct the evolutionary history of inflammatory pathway development and adaptation to pathogens.

• Translation-Focused Approaches: Employing recombinant rhesus IL1A alongside human IL1A in preclinical studies helps predict translational challenges in therapeutic development targeting the IL-1 pathway.

The value of such comparative approaches was demonstrated in the study by Villinger et al., which established important foundations for understanding cytokine evolution across primate species . Similar approaches can be expanded to create comprehensive frameworks for understanding species-specific immune responses, with important implications for both evolutionary immunology and translational research.