Recombinant Rat C-C motif chemokine 3 protein (Ccl3)

What is Recombinant Rat C-C motif chemokine 3 protein (Ccl3) and what are its key biological functions?

Recombinant Rat C-C motif chemokine 3 protein (Ccl3), also known as Macrophage Inflammatory Protein 1 alpha (MIP-1α), is a full-length protein spanning amino acids 24 to 92 when expressed in recombinant systems. It functions primarily as a monokine with both inflammatory and chemokinetic properties. Ccl3 mediates its effects by binding to multiple chemokine receptors, specifically CCR1, CCR4, and CCR5. In immunological contexts, it serves as one of the major HIV-suppressive factors produced by CD8+ T-cells, with recombinant MIP-1-alpha demonstrating dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) .

From a structural perspective, Ccl3 belongs to the intercrine beta family, also known as the chemokine CC family. The protein exhibits specific amino acid sequence (A P Y G A D T P T A C C F S Y G R Q I P R K F I A D Y F E T S S L C S Q P G V I F L T K R N R Q I C A D P K E T W V Q E Y I T E L E L N A) that confers its functional capabilities . Importantly, Ccl3 plays a regulatory role in normal hematopoiesis, particularly in promoting myeloid lineage differentiation and maintaining the size of the hematopoietic stem and progenitor cell (HSPC) pool .

How can I verify the activity of recombinant Rat Ccl3 protein in my experimental system?

Verification of recombinant Rat Ccl3 activity can be accomplished through multiple complementary methods, each providing distinct insights into protein functionality. The most established approach is through chemotaxis assays, where Ccl3 demonstrates dose-dependent chemotactic activity. For example, recombinant Rat Ccl3/MIP-1α induces chemotaxis in BaF3 mouse pro-B cell lines transfected with human CCR5 receptors. This activity follows a dose-dependent pattern that can be quantitatively measured and used as a verification standard .

For robust validation, researchers should implement neutralization experiments using specific antibodies. Mouse Anti-Rat Ccl3/MIP-1α Monoclonal Antibody can effectively neutralize the chemotactic activity of recombinant Rat Ccl3/MIP-1α. The neutralization dose (ND₅₀) typically ranges from 0.075-0.45 μg/mL, providing a quantitative metric for antibody efficacy . Additionally, Western blot analysis can confirm protein expression in relevant experimental systems. For instance, Western blots of NR8383 rat alveolar macrophage cell line lysates, when treated with LPS (10 μg/mL for 4 hours), demonstrate specific Ccl3/MIP-1α bands at approximately 12 kDa under reducing conditions .

What are the alternative names for Ccl3 and how do they relate to its biological classification?

Rat C-C motif chemokine 3 protein is known by multiple nomenclatures in scientific literature, reflecting its discovery context and functional characterization. The primary alternative designation is Macrophage Inflammatory Protein 1-alpha (MIP-1-alpha), which references its historical identification as a protein produced by macrophages with inflammatory properties. Additional recognized aliases include Mip1a, Scya3, Small-inducible cytokine A3, and C-C motif chemokine 3 .

From a taxonomic perspective, Ccl3 belongs to the intercrine beta family, more commonly referred to as the chemokine CC family. This classification is based on structural characteristics, particularly the arrangement of conserved cysteine residues. The systematic organization of chemokines places Ccl3 within a broader family of related molecules that share structural similarities and often exhibit overlapping yet distinct functions . Understanding these nomenclature variations is essential for comprehensive literature searches and for placing research findings in proper context within the field of chemokine biology.

What are the optimal expression systems for producing high-quality recombinant Rat Ccl3?

Escherichia coli represents the predominant expression system for producing high-quality recombinant Rat Ccl3 protein suitable for research applications. When properly optimized, E. coli expression systems yield Rat Ccl3 with >95% purity and endotoxin levels below 1 EU/μg, making the protein appropriate for a wide range of experimental applications including SDS-PAGE and functional studies . This bacterial expression system offers several advantages including cost-effectiveness, scalability, and established purification protocols.

When working with E. coli-expressed Ccl3, researchers should implement rigorous quality control measures. Specifically, SDS-PAGE analysis under both reducing and non-reducing conditions should be performed to verify protein integrity and purity. Visual confirmation through Western blotting using specific anti-Ccl3 antibodies provides additional validation . For functional verification, chemotaxis assays using CCR5-transfected cell lines represent the gold standard, as they directly measure the biological activity of the recombinant protein . Researchers should note that recombinant Ccl3 is an active protein capable of eliciting biological responses in vivo, necessitating appropriate handling precautions throughout the experimental workflow .

What techniques are most effective for detecting endogenous and recombinant Rat Ccl3 expression in experimental models?

Multiple complementary techniques provide robust detection of both endogenous and recombinant Rat Ccl3 expression across experimental systems. Western blotting represents a primary approach, particularly effective when analyzing NR8383 rat alveolar macrophage cell lines following LPS stimulation (10 μg/mL for 4 hours). Under these conditions, probing PVDF membranes with 2 μg/mL of Mouse Anti-Rat Ccl3/MIP-1α Monoclonal Antibody followed by HRP-conjugated secondary antibody detection reveals specific bands at approximately 12 kDa under reducing conditions .

For quantitative assessment of Ccl3 protein levels in tissue samples, enzyme-linked immunosorbent assay (ELISA) provides superior sensitivity. When analyzing cardiac tissue, for example, samples should be processed in solutions containing protease inhibitors, followed by centrifugation to obtain supernatants suitable for ELISA analysis . This approach enables precise quantification of both endogenous and experimentally induced Ccl3 expression levels.

For gene expression studies, RT-PCR using high-fidelity platinum Taq polymerase represents an effective strategy for amplifying Ccl3 mRNA from infected tissues. This technique proves particularly valuable when cloning Ccl3 genes lacking signal sequences for subsequent expression vector construction . Collectively, these methodological approaches provide complementary data on Ccl3 expression at both protein and transcript levels.

How can I design experiments to study Ccl3 receptor binding specificity and downstream signaling?

Designing robust experiments to investigate Ccl3 receptor binding specificity and downstream signaling requires a multi-faceted approach combining cellular, molecular, and pharmacological techniques. The primary receptors for Ccl3—CCR1, CCR4, and CCR5—exhibit differential binding characteristics that can be experimentally distinguished . Begin by establishing cell models expressing individual receptor subtypes, such as BaF3 mouse pro-B cell lines transfected with human CCR5, which provide clean systems for receptor-specific analyses .

Chemotaxis assays represent the gold standard for functional assessment of receptor engagement. These assays should be performed with increasing concentrations of recombinant Rat Ccl3 (typically ranging from 1-100 ng/mL) to establish dose-response relationships. For receptor specificity determination, implement competitive binding experiments using receptor-selective antagonists. For instance, MetRANTES functions as a powerful tool in this context, as it specifically antagonizes CCR1 and CCR5 receptors . Daily treatment with 150 μg of MetRANTES can effectively block Ccl3-mediated responses in vivo .

For downstream signaling analysis, phosphorylation status of key intermediates should be assessed by Western blotting following Ccl3 stimulation at various time points. Additionally, neutralization experiments using specific antibodies against Ccl3 provide critical mechanistic insights. The neutralization dose (ND₅₀) typically ranges from 0.075-0.45 μg/mL for antibodies such as Mouse Anti-Rat Ccl3/MIP-1α Monoclonal Antibody . These methodologies collectively enable comprehensive characterization of receptor-mediated Ccl3 signaling cascades.

How does Ccl3 influence hematopoietic stem and progenitor cell (HSPC) differentiation and what are the implications for studying hematologic disorders?

Ccl3 exerts profound regulatory influence on hematopoietic stem and progenitor cell (HSPC) dynamics, with particularly significant effects on myeloid lineage differentiation. Genetic ablation studies using CCL3⁻/⁻ mice have revealed that Ccl3 deficiency results in a distinct hematopoietic phenotype characterized by diminished mature myeloid populations concurrent with increased myeloid progenitors and expanded HSPC pools . This paradoxical finding indicates that Ccl3 functions as a positive regulator of terminal myeloid differentiation while simultaneously controlling HSPC population size.

Importantly, these effects appear independent of the supportive bone marrow microenvironment (BMME), suggesting direct effects on hematopoietic cells rather than indirect influence through niche modulation . This mechanism represents a previously unrecognized role of Ccl3 in maintaining homeostatic hematopoiesis with significant implications for hematologic malignancy research. In chronic myelogenous leukemia (CML) models, Ccl3 has been identified as critical for leukemia stem cell population maintenance and disease progression .

What is the role of Ccl3 in infectious disease models and how can it be manipulated experimentally?

Ccl3 demonstrates significant involvement in infectious disease pathophysiology, particularly in models of Trypanosoma cruzi infection. Experimental approaches to study and manipulate Ccl3 in these contexts include genetic, vaccination, and pharmacological strategies. For genetic manipulation, cloning of Rat Ccl3/MIP-1α from myocardial samples of acutely T. cruzi-infected rats can be accomplished using RT-PCR with high-fidelity platinum Taq polymerase . During this process, it's critical to identify and modify the signal peptide-encoding sequence to optimize secretion characteristics in subsequent expression systems.

DNA vaccination represents an innovative approach to modulate Ccl3 activity in vivo. Construction of vaccination plasmids incorporates Ccl3 genes into vectors such as VR2001-TOPO DNA plasmid, which contains appropriate secretion signal sequences. These constructs should be propagated in bacterial culture, purified using appropriate maxiprep kits, and extensively washed with ultrapure water using systems like Amicon-100 prior to administration .

Pharmacological modulation offers precise temporal control over Ccl3 signaling. MetRANTES, a modified form of human CCL5/RANTES, functions as a potent antagonist of CCR1 and CCR5 receptors—the primary receptors for Ccl3. In rat models, subcutaneous administration of 150 μg MetRANTES daily effectively blocks Ccl3-mediated responses . Assessment of intervention efficacy should incorporate tissue-specific ELISA for cytokine/chemokine quantification alongside histopathological analysis to measure inflammation and parasitism in affected tissues .

How can conflicting findings on Ccl3 function be reconciled through improved experimental design?

Reconciling conflicting findings regarding Ccl3 function requires sophisticated experimental design addressing the multifaceted nature of chemokine biology. A primary consideration is the context-dependent nature of Ccl3 activity. For example, while Ccl3 promotes myeloid differentiation in normal hematopoiesis, it also contributes to leukemia stem cell maintenance in malignant contexts . This apparent contradiction can be resolved through careful comparison of microenvironmental factors in normal versus malignant states.

Experimental approaches should incorporate both loss-of-function and gain-of-function strategies. Studies utilizing CCL3⁻/⁻ mice provide valuable insights into the consequences of complete Ccl3 deficiency , while targeted overexpression models using vaccination plasmids like VR2001-Ccl3 illuminate the effects of enhanced Ccl3 signaling . Comparative analysis between these models enables identification of dose-dependent effects that may explain seemingly contradictory outcomes.

Temporal considerations are equally critical, as Ccl3 effects may vary during different developmental stages or disease progression phases. Implementing inducible systems for Ccl3 manipulation allows precise control over timing, revealing stage-specific functions. Additionally, receptor-specific approaches using selective antagonists like MetRANTES help dissect the contributions of different Ccl3 receptors (CCR1, CCR4, and CCR5) to observed phenotypes. Finally, comprehensive assessment of both direct and indirect Ccl3 effects through analysis of multiple parameters—including cytokine profiles, cellular composition, and functional outcomes—provides the integrated perspective necessary to resolve apparent contradictions in Ccl3 biology.

How does Rat Ccl3 compare structurally and functionally to human and mouse orthologs?

Rat Ccl3 shares substantial structural and functional homology with its human and mouse orthologs, though species-specific variations exist with implications for translational research. Structurally, Rat Ccl3 is expressed as a full-length protein spanning amino acids 24 to 92 when produced in recombinant systems . The protein belongs to the intercrine beta (chemokine CC) family across all three species, characterized by conserved cysteine residue patterns that define secondary structure elements essential for receptor binding.

Functionally, Rat Ccl3 mirrors its orthologs in binding to multiple chemokine receptors—specifically CCR1, CCR4, and CCR5—though binding affinities may vary slightly between species . The capacity to induce chemotaxis represents a conserved function across species, with Rat Ccl3 demonstrating chemotactic activity in appropriate receptor-expressing cell lines . All three orthologs function as HIV-suppressive factors, with recombinant proteins showing dose-dependent inhibition of various HIV strains and related viruses .

In experimental contexts, it's critical to note that while functional conservation exists, cross-species reactivity is not guaranteed. For instance, antibodies developed against Rat Ccl3 may not recognize human or mouse proteins with equal efficiency. This consideration necessitates species-appropriate reagents for detection and neutralization studies. When using model systems for translational research, researchers should acknowledge these species-specific variations when extrapolating findings to human applications.

What are the most significant technical challenges in working with recombinant Rat Ccl3 and how can they be overcome?

Researchers working with recombinant Rat Ccl3 face several technical challenges that require specific strategies to maintain experimental integrity. Protein stability represents a primary concern, as Ccl3 can undergo degradation during storage and handling. To mitigate this, recombinant preparations should be stored in small aliquots at -80°C to minimize freeze-thaw cycles, and working solutions should include appropriate protease inhibitors, particularly when processing tissue samples for subsequent analysis .

Endotoxin contamination presents another significant challenge, especially when using E. coli expression systems. High-quality preparations should maintain endotoxin levels below 1 EU/μg , achievable through rigorous purification protocols and endotoxin testing prior to experimental use. This consideration becomes particularly critical for in vivo applications and cellular assays where endotoxin can confound results by independently activating inflammatory pathways.

How should researchers interpret conflicting data on Ccl3 function in different experimental models?

Interpreting conflicting data on Ccl3 function across experimental models requires systematic analysis of multiple variables that influence chemokine activity. First, researchers must evaluate context-dependent factors—Ccl3 demonstrates distinctive effects in different tissue microenvironments and disease states. For instance, while Ccl3 promotes myeloid differentiation under normal conditions, it also contributes to leukemia stem cell maintenance in malignant settings . These seemingly contradictory findings likely reflect the integrated response of complex biological systems to Ccl3 signaling rather than true mechanistic inconsistencies.

Methodological variations substantially impact observed outcomes. Differences in dosing regimens, administration routes, and timing of interventions may yield divergent results. When comparing studies using genetic models (CCL3⁻/⁻ mice) versus pharmacological approaches (MetRANTES administration), distinctions between acute inhibition and developmental compensation in knockout models must be considered . Furthermore, receptor expression patterns vary across cell types and experimental systems—Ccl3 signals through multiple receptors (CCR1, CCR4, and CCR5) with potentially different downstream effects .

To reconcile conflicting findings, researchers should implement comprehensive experimental designs incorporating multiple complementary approaches. This includes assessing Ccl3 function across different concentrations, time points, and cellular contexts while simultaneously evaluating receptor expression and signaling pathway activation. Additionally, examining both physiological and pathological states within the same experimental system provides critical comparative data that can resolve apparent contradictions in Ccl3 biology.

What evidence supports targeting Ccl3 signaling in treatment strategies for hematologic malignancies?

Compelling evidence positions Ccl3 as a potential therapeutic target in hematologic malignancies, particularly chronic myelogenous leukemia (CML). Mechanistic studies have identified Ccl3 as a critical factor for maintaining leukemia stem cell populations and supporting disease progression in murine CML models . This finding aligns with observations of Ccl3 overexpression across multiple malignancies, where it contributes to microenvironmental dysfunction that promotes cancer cell survival and proliferation.

Ccl3's dual role in regulating both normal and malignant hematopoiesis provides the theoretical foundation for therapeutic intervention. In normal hematopoiesis, Ccl3 promotes myeloid lineage differentiation while controlling the size of the hematopoietic stem and progenitor cell (HSPC) pool . This function appears independent of the bone marrow microenvironment, suggesting direct effects on hematopoietic cells rather than indirect niche modulation. In malignant contexts, disruption of this regulatory function contributes to abnormal myeloid development and expansion of undifferentiated cells.

Targeting strategies must account for Ccl3's physiological importance in normal hematopoiesis. Complete inhibition might adversely affect normal myeloid development, suggesting that therapeutic approaches may require careful titration or contextual specificity . Potential targeting approaches include receptor antagonists similar to MetRANTES (which blocks CCR1 and CCR5) , neutralizing antibodies, or small molecule inhibitors of downstream signaling pathways. The evolving understanding of Ccl3 biology in both normal and malignant hematopoiesis continues to refine these potential therapeutic applications.

How can recombinant Ccl3 and related tools be utilized in developing vaccination strategies against infectious diseases?

Recombinant Ccl3 and associated molecular tools offer innovative approaches for vaccination strategies against infectious diseases, particularly Trypanosoma cruzi infections. DNA vaccination represents a frontier application, where Ccl3-encoding plasmids modulate immune responses to enhance protection. For constructing effective vaccination plasmids, researchers should clone the Ccl3 gene using RT-PCR with high-fidelity platinum Taq polymerase from relevant tissue samples, such as myocardial specimens from acutely infected animals .

When designing these constructs, careful attention to signal peptide sequences optimizes secretion characteristics. Vaccination vectors like VR2001-TOPO DNA plasmid already contain appropriate secretion signal sequences, necessitating modification of the native Ccl3 gene to remove its endogenous signal sequence . Following construction, plasmids should undergo sequence verification to ensure correct orientation and reading frame alignment with the vector's signal peptide sequence.

For vaccine preparation, plasmids should be propagated in large-scale bacterial cultures (1L of Luria broth with appropriate antibiotics), purified using maxiprep kits, and extensively washed with ultrapure water using systems like Amicon-100 . Efficacy assessment should incorporate comprehensive immunological analysis including cytokine/chemokine profiling through ELISA, histopathological examination of affected tissues, and functional protection assays. This approach leverages Ccl3's immunomodulatory properties to enhance vaccine-induced protection against challenging infectious agents.

What are the emerging research directions for understanding Ccl3 biology in novel therapeutic contexts?

Emerging research directions for Ccl3 biology encompass several innovative therapeutic contexts that extend beyond traditional applications. First, the complex interplay between Ccl3 and the bone marrow microenvironment in both normal and malignant hematopoiesis represents a frontier area. While current evidence suggests Ccl3 regulates hematopoietic stem cell function independent of microenvironmental changes , deeper investigation of potential bidirectional interactions may reveal novel therapeutic opportunities, particularly in conditions where microenvironmental dysfunction contributes to pathology.

Second, combinatorial approaches pairing Ccl3 modulation with established therapies warrant exploration. For instance, in hematologic malignancies, targeting Ccl3 signaling might sensitize leukemia stem cells to conventional chemotherapeutics or tyrosine kinase inhibitors. Similarly, in infectious disease contexts, coordinating Ccl3-based vaccination strategies with traditional antimicrobial approaches could enhance efficacy through complementary mechanisms of action .

Third, advanced delivery systems for Ccl3-targeting therapeutics represent an important technological frontier. Nanoparticle-based delivery of Ccl3 antagonists or expression constructs could improve pharmacokinetics and reduce off-target effects. Similarly, cell-based delivery systems using engineered immune cells that secrete modified Ccl3 variants might enable precise spatial and temporal control over Ccl3 activity in specific microenvironments. These emerging directions collectively expand the potential therapeutic applications of Ccl3 biology beyond current paradigms.