Recombinant Rat C-C motif chemokine 7 protein (Ccl7) (Active)

What is Recombinant Rat C-C Motif Chemokine 7 protein (CCL7)?

Recombinant Rat CCL7 (also known as Monocyte Chemoattractant Protein 3 or MCP-3) is a full-length protein produced through E. coli expression systems. The protein functions as a chemoattractant, primarily for monocytes, and belongs to the C-C chemokine family. The mature protein has a molecular weight of approximately 8.5 kDa and consists of 74 amino acids (residues 24-97 of the full sequence) . CCL7 serves multiple roles in immune regulation, particularly in recruiting inflammatory cells to sites of injury or infection. Its complete amino acid sequence is QPDGTNSST CCYVKKQKIP KRNLKSYRKI TSSRCPWEAV IFKTKKGMEV CAEAHQKWVE EAIAYLDMKT STPKP .

What are the optimal storage and reconstitution protocols for Recombinant Rat CCL7?

For optimal stability and activity, Recombinant Rat CCL7 should be stored at -20°C to -80°C immediately upon receipt. The protein is typically supplied as a lyophilized powder from a PBS buffer (pH 7.4) solution that has been filtered through a 0.2 μm filter . When reconstituting, the vial should be briefly centrifuged prior to opening to bring contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration between 0.1-1.0 mg/mL . For long-term storage, addition of glycerol (5-50% final concentration, with 50% being standard) is recommended, followed by aliquoting to avoid repeated freeze-thaw cycles which significantly diminish protein activity . Working aliquots can be prepared at appropriate concentrations based on experimental requirements.

How is the biological activity of Recombinant Rat CCL7 determined?

The biological activity of Recombinant Rat CCL7 is primarily determined through a chemotaxis bioassay using human monocytes. The protein is considered fully biologically active when it induces chemotaxis in a concentration range of 10-100 ng/ml . This functional assay directly measures the protein's ability to stimulate directional migration of target cells, which is its primary biological function. Additional quality control parameters include purity assessment via SDS-PAGE and HPLC (>95% purity) and endotoxin testing using the LAL method (<1.0 EU/μg) . These parameters ensure that experimental results reflect the protein's true biological activity rather than contaminants or degradation products.

What methodological considerations are important when studying CCL7's role in cancer immunology?

Methodologically, researchers should:

• Employ appropriate animal models: Studies have utilized Kras^LSL−G12D/+p53^fl/fl (KP) and Kras^LSL−G12D/+Lkb1^fl/fl (KL) NSCLC mouse models to investigate CCL7's effects .

• Consider viral delivery methods: Lentiviral vectors expressing CCL7 (Lenti-CCL7) have been used for intranasal administration in mouse models to study therapeutic effects .

• Analyze multiple immune cell populations: CCL7 significantly promotes cDC1 infiltration, which subsequently enhances CD8+ T cell responses. Flow cytometry should assess changes in cDC1 (CD11c+XCR1+) and CD8+IFNγ+ T cells in tumor-draining lymph nodes and lung-infiltrating lymphocytes .

• Evaluate combined immunotherapy approaches: CCL7 has been shown to enhance the efficacy of anti-PD-1 checkpoint immunotherapy, especially in models resistant to anti-PD-1 monotherapy . Experimental designs should incorporate combination therapies to fully evaluate CCL7's potential.

• Incorporate immunohistochemistry: IHC analysis should be used to confirm CCL7 expression and to quantify changes in CD11c, XCR1, and CD8 staining in tumor tissues .

How does CCL7 contribute to neurological pain mechanisms and what experimental approaches are used to study this?

CCL7 plays a significant role in trigeminal neuropathic pain through specific neuroimmune mechanisms. Recent research using the partial infraorbital nerve transection (pIONT) model has revealed important insights into these pathways .

Key experimental approaches include:

• Neuropathic pain models: The pIONT model induces persistent mechanical allodynia starting from day 3 and lasting more than 21 days post-operation, providing a reliable model for studying CCL7's role in pain mechanisms .

• Gene expression analysis: Quantitative PCR (qPCR) has demonstrated that Ccl7 mRNA is significantly upregulated in the trigeminal ganglion (TG) at days 1, 3, 10, and 21 after pIONT, indicating sustained involvement throughout the pain development and maintenance phases .

• Cell-specific localization: Immunofluorescence double-staining with markers for neurons (TUJ1), satellite cells (GS), and macrophages (IBA-1) reveals that CCL7 is predominantly expressed in TG neurons rather than glial or immune cells . Further characterization using neuronal subtype markers (NF200, IB4, CGRP) shows that CCL7 is distributed across multiple neuron types but primarily colocalizes with NF200-positive myelinated neurons .

• Functional manipulation: RNA interference using Ccl7 siRNA injection into the TG significantly reduces pain behaviors when administered after pIONT, providing direct evidence for CCL7's contribution to neuropathic pain maintenance .

• Signaling pathway analysis: CCL7 activates ERK in TG neurons via CCR2 and CCR3 receptors, enhancing neuronal excitability that contributes to the maintenance of trigeminal neuropathic pain .

What are the important considerations for structural and functional studies of recombinant chemokines like CCL7?

Structural and functional studies of recombinant chemokines require careful consideration of several factors that can impact experimental outcomes and interpretations. While the search results don't provide specific structural information about CCL7, we can draw parallels from related research on recombinant proteins :

• Expression systems: The choice between prokaryotic (E. coli) and eukaryotic expression systems significantly affects post-translational modifications. For CCL7, E. coli expression systems are commonly used , but researchers should recognize the absence of glycosylation and other eukaryotic modifications that might alter protein folding or function.

• Protein tagging strategies: Tags can facilitate purification but may interfere with protein function. Tag-free recombinant CCL7 is optimal for functional studies to avoid potential interference with receptor binding or oligomerization .

• Species-specific variations: Significant variation exists in the functional and pharmacological properties of chemokines from different species. When extrapolating from rat CCL7 to human applications, these differences must be considered. The complete sequence of rat CCL7 (QPDGTNSSTC CYVKKQKIPK RNLKSYRKIT SSRCPWEAVI FKTKKGMEVC AEAHQKWVEE AIAYLDMKTS TPKP) should be compared with human CCL7 to identify conserved and divergent regions that might affect receptor binding and function .

• Oligomerization state: Many chemokines form dimers or higher-order oligomers that affect their function. Analytical techniques such as size exclusion chromatography should be employed to characterize the oligomeric state of recombinant CCL7 under experimental conditions.

• Receptor binding studies: CCL7 interacts with multiple chemokine receptors, including CCR2 and CCR3 . Binding assays with purified receptors or receptor-expressing cell lines are essential to characterize these interactions and their functional consequences.

What control experiments are essential when studying CCL7-mediated chemotaxis?

When designing chemotaxis experiments using recombinant rat CCL7, several control experiments are crucial for proper interpretation of results:

• Concentration gradient controls: Since the biologically active concentration range for CCL7 in chemotaxis assays is 10-100 ng/ml , multiple concentrations within and beyond this range should be tested to establish a dose-response curve. Both sub-threshold and saturating concentrations provide important information about sensitivity and receptor saturation.

• Specificity controls:

  • Receptor antagonists: Specific antagonists for CCR2 and CCR3 (the primary receptors for CCL7 ) should be included to confirm receptor-specific responses.

  • Blocking antibodies: Anti-CCL7 neutralizing antibodies help verify that observed effects are specifically due to CCL7 activity.

  • Heat-inactivated protein: Denatured CCL7 controls for non-specific protein effects.

• Cell-type specificity: While monocytes are primary targets of CCL7, testing multiple immune cell populations (dendritic cells, T cells, neutrophils) can provide valuable information about differential responsiveness. This is particularly important when investigating CCL7's role in cancer immunity where it recruits cDC1 .

• Chemokinesis versus chemotaxis: Checkerboard assays (where equal concentrations of chemokine are placed in both upper and lower chambers) distinguish between directional migration (chemotaxis) and random migration (chemokinesis).

• Positive controls: Including well-characterized chemokines (e.g., CCL2/MCP-1 for monocytes) provides benchmarks for comparing CCL7 activity and verifies assay functionality.

How can researchers effectively study CCL7's role in complex disease models?

Studying CCL7 in complex disease models requires integrated experimental approaches that capture both molecular mechanisms and physiological outcomes:

What are the most common issues encountered when working with recombinant CCL7 and how can they be addressed?

Researchers working with recombinant CCL7 frequently encounter several technical challenges that can affect experimental outcomes:

• Activity loss during storage/handling:

  • Problem: Freeze-thaw cycles significantly reduce protein activity.

  • Solution: Store as recommended at -20°C/-80°C in small aliquots with 5-50% glycerol . Avoid multiple freeze-thaw cycles.

• Inconsistent chemotaxis results:

  • Problem: Variable cell migration responses between experiments.

  • Solutions:

    • Standardize monocyte isolation procedures

    • Use freshly reconstituted CCL7 at precise concentrations

    • Ensure consistent 10-100 ng/ml working concentrations

    • Verify proper formation of concentration gradients in migration chambers

• Endotoxin contamination:

  • Problem: Bacterial endotoxins in recombinant preparations can activate immune cells independently of CCL7.

  • Solution: Verify endotoxin levels are below 1.0 EU/μg using the LAL method . Consider additional endotoxin removal steps if necessary.

• Species cross-reactivity issues:

  • Problem: Rat CCL7 may have different potency on human versus rodent cells.

  • Solution: Validate activity on species-matched cells before cross-species experiments. Consider species-specific differences in receptor binding affinity.

• Protein aggregation:

  • Problem: Improper reconstitution can lead to protein aggregation and reduced activity.

  • Solution: Follow precise reconstitution protocols, including centrifuging the vial before opening and reconstituting in deionized sterile water . Filter solutions if aggregates are observed.

How can researchers distinguish between CCL7-specific effects and other chemokine activities in complex systems?

Distinguishing CCL7-specific effects from those of other chemokines presents a significant challenge due to redundancy in the chemokine system. Researchers can employ these approaches:

• Receptor profile analysis:

  • CCL7 signals through multiple receptors including CCR2 and CCR3

  • Map the receptor expression profile of target cells (flow cytometry or RT-PCR)

  • Use receptor-specific antagonists to isolate contributions of individual signaling pathways

• Genetic approaches:

  • RNA interference targeting CCL7 specifically (as demonstrated in trigeminal ganglion studies )

  • CRISPR/Cas9-mediated knockout of CCL7 or its receptors

  • Conditional knockout models to achieve temporal and spatial specificity

• Neutralization strategies:

  • CCL7-specific neutralizing antibodies

  • Soluble decoy receptors

  • Custom-designed receptor antagonists

• Comparative studies:

  • Side-by-side comparison with related chemokines (CCL2/MCP-1, CCL8/MCP-2)

  • Concentration-matched experiments to account for potency differences

  • Checkerboard analyses to distinguish specific receptor-ligand interactions

• Chimeric proteins:

  • Domain-swapping between CCL7 and related chemokines

  • Structure-function studies to identify unique functional domains

  • Tagged variants to track localization while maintaining function

What are promising research areas for extending our understanding of CCL7 biology?

Several emerging areas show particular promise for advancing CCL7 research:

• Cancer immunotherapy applications:

  • CCL7's ability to recruit cDC1 and enhance anti-PD-1 checkpoint immunotherapy efficacy warrants further investigation in multiple cancer types beyond NSCLC

  • Exploration of CCL7 as an adjuvant for other immunotherapies

  • Development of CCL7-based therapeutic delivery systems

  • Investigation of CCL7 as a biomarker for immunotherapy response prediction

• Neuroimmune interactions:

  • Further characterization of CCL7's role in neuron-immune cell crosstalk in pain conditions

  • Extended studies of CCL7 in other neurological disorders

  • Investigation of CCL7's modulatory effects on synaptic plasticity and neural circuit function

  • Development of CCL7-targeting analgesics

• Structural biology approaches:

  • High-resolution structural studies of CCL7-receptor complexes

  • Structure-based design of selective CCL7 modulators

  • Investigation of CCL7 oligomerization dynamics and their functional significance

  • Comparative structural analysis across species

• Systems biology integration:

  • Mapping the complete CCL7 interactome

  • Network analysis of CCL7 signaling in different physiological contexts

  • Multi-omics approaches to comprehensively characterize CCL7-mediated responses

  • Computational modeling of CCL7 gradient formation and cellular responses

• Translational applications:

  • Development of CCL7-based diagnostic tools

  • Design of CCL7 mimetics with enhanced stability or receptor selectivity

  • Exploration of CCL7 as a vaccine adjuvant

  • Investigation of CCL7 polymorphisms in disease susceptibility