Recombinant Pig C-C motif chemokine 4 (CCL4)

What is recombinant pig CCL4 and how does it differ from native CCL4?

Recombinant pig CCL4 (Sus scrofa) is a laboratory-engineered version of the native chemokine produced through genetic engineering. The recombinant form is typically derived from expression systems like E. coli, with the gene encoding for pig CCL4 (accession number Q711P4) inserted into the host organism for expression . Unlike native CCL4 isolated from pig tissues, recombinant CCL4 offers precise control over modifications, large-scale synthesis capabilities, and consistent batch-to-batch quality. The recombinant protein usually contains the expressed region from Ala24-Asn92 of the full-length protein and may include additional tags (commonly His-tag at the N-terminus) to facilitate purification and detection . While native CCL4 contains post-translational modifications specific to porcine cells, recombinant versions may lack these modifications depending on the expression system used.

What expression systems are commonly used for producing recombinant pig CCL4?

• E. coli expression: Provides high protein yields but lacks eukaryotic post-translational modifications. Typically produces protein in inclusion bodies requiring refolding protocols.

• Mammalian expression systems: Yield lower quantities but provide proper folding and post-translational modifications that may be crucial for certain functional studies.

• Insect cell systems: Offer a middle ground between bacterial and mammalian systems regarding yield and post-translational modifications.

• Yeast expression systems: Can provide higher yields than mammalian cells with some post-translational capabilities.

For studies focused on structural analyses or applications where post-translational modifications are not critical, E. coli-derived recombinant pig CCL4 with >95% purity (as determined by SDS-PAGE) is generally sufficient . For functional assays investigating receptor interactions or signaling pathways, researchers might consider mammalian expression systems to maintain native-like conformations.

What are the optimal storage conditions for maintaining recombinant pig CCL4 activity?

For optimal stability and activity retention of recombinant pig CCL4, researchers should follow these evidence-based storage protocols:

• Short-term storage: Maintain at -20°C in aliquots to avoid repeated freeze-thaw cycles .

• Working solution preparation: Reconstitute lyophilized protein in sterile buffer (typically phosphate-buffered solutions at pH 7.2-7.4).

• Formulation considerations: Commercial preparations are commonly supplied as filtered solutions in buffers containing sodium phosphate (approximately 50 mM Na₂HPO₄, pH 7.4), sodium chloride (0.5 M NaCl), and stabilizers like imidazole (80 mM) .

• Avoiding protein degradation: Add protease inhibitors if working with the protein for extended periods at temperatures above freezing.

• Activity assessment: Before experimental use, verify protein activity through functional assays specific to chemokine activity (chemotaxis assays or receptor binding assays).

Studies have shown that recombinant proteins like CCL4 can lose up to 30% activity after three freeze-thaw cycles, emphasizing the importance of proper aliquoting upon initial reconstitution.

What are the recommended protocols for using recombinant pig CCL4 in T-cell expansion studies?

When designing experiments to investigate T-cell expansion using recombinant pig CCL4, researchers should implement the following methodological approaches:

• Isolation of porcine peripheral blood mononuclear cells (PBMCs): Use density gradient centrifugation with Ficoll-Paque followed by washing with PBS containing 2% FBS.

• T-cell subset analysis: Employ flow cytometry with antibodies specific for CD4+, CD8+, and CD4+/CD8+ double-positive T cells to establish baseline measurements before CCL4 treatment .

• CCL4 treatment dosage optimization: Typically, concentrations ranging from 10-500 ng/mL should be tested, with 100 ng/mL serving as a standard starting point for most applications.

• Time-course analyses: Measure T-cell expansion at multiple timepoints (e.g., 24, 48, 72 hours post-treatment) to capture both early and sustained responses .

• Functional verification: Complement proliferation assays with IFN-γ ELISPOT to quantify the number of IFN-γ secreting cells per million PBMCs (a range of 2-200 IFN-γ secreting cells per million PBMCs has been observed in immunized pigs) .

In a comparative study examining T-cell ratios, researchers documented that CD4+, CD8+, and CD4+/CD8+ double-positive T cell proportions showed significant variation at different timepoints post-immunization (day 0, 28, and 35), with CD8+ and CD4+/CD8+ DP T cells demonstrating the most pronounced expansion in some experimental subjects .

How can recombinant pig CCL4 be effectively used in viral infection models?

Recombinant pig CCL4 has demonstrated valuable applications in viral infection studies, particularly for investigating host immune responses. Researchers should consider the following methodological approach:

• Viral challenge model selection: Choose appropriate viral models relevant to swine health, such as African Swine Fever Virus (ASFV) or other porcine viruses .

• In vitro infection protocol:

  • Establish cell culture systems using porcine macrophages or suitable cell lines

  • Determine optimal multiplicity of infection (MOI) through pilot studies (MOIs of 1 and 5 have been used successfully)

  • Monitor viral replication through RT-qPCR for viral genes and Western blot for viral proteins

• CCL4 treatment regimens:

  • Pre-infection treatment: Add recombinant CCL4 (50-200 ng/mL) 6-24 hours before viral challenge

  • Post-infection treatment: Add CCL4 at various timepoints after infection to assess intervention potential

  • Continuous exposure: Maintain CCL4 in culture medium throughout the experimental timeline

• Readouts for assessing antiviral effects:

  • Viral load quantification by qPCR

  • Viral protein expression by Western blot or flow cytometry

  • Cell viability assays (MTT or CCK-8) to differentiate between antiviral effects and cytotoxicity

  • Cytokine/chemokine profiling to characterize the broader immune response

Research has demonstrated that CCL4 can inhibit viral replication in certain models, with significant downregulation of viral gene expression observed between 6 and 36 hours post-infection .

What controls should be included when evaluating recombinant pig CCL4 functionality in immunological assays?

Proper experimental controls are essential for rigorous evaluation of recombinant pig CCL4 functionality. Researchers should implement the following control strategies:

• Positive biological activity controls:

  • Known chemotactic factors (e.g., IL-8 for neutrophils)

  • Commercially validated recombinant pig CCL4 from established vendors

  • Species-matched native CCL4 (when available)

• Negative controls:

  • Heat-inactivated recombinant CCL4 (95°C for 10 minutes)

  • Irrelevant recombinant protein expressed in the same system

  • Buffer-only treatments matching the CCL4 formulation buffer

• Specificity controls:

  • CCL4 receptor antagonists to confirm receptor-dependent effects

  • Anti-CCL4 neutralizing antibodies to block activity

  • Competitive inhibition using excessive amounts of unlabeled CCL4

• Experimental validation controls:

  • Dose-response curves (10-500 ng/mL) to establish optimal concentration

  • Time-course studies (6-72 hours) to determine temporal dynamics

  • Cross-species comparisons when appropriate (e.g., human vs. porcine CCL4)

Inclusion of these controls helps distinguish between specific CCL4-mediated effects and non-specific responses or contaminant effects, particularly when evaluating immune cell functions like chemotaxis, activation, or cytokine production.

How does recombinant pig CCL4 interact with glucose metabolism in immune cells?

Recent research has uncovered a critical relationship between CCL4 and cellular metabolism, particularly glucose utilization pathways. When investigating this interaction, researchers should consider:

• Metabolic assessment methodology:

  • Glucose consumption assays using fluorescent glucose analogs

  • Lactate production measurement as an indicator of glycolytic activity

  • Seahorse XF analysis to determine glycolytic rates and mitochondrial respiration

  • Expression analysis of key glycolytic enzymes (HK2, PKM2, LDHA)

• Experimental design considerations:

  • Compare high glucose (25 mM) versus low glucose (5.5 mM) culture conditions

  • Utilize glycolytic inhibitors like 2-deoxy-D-glucose (2-DG) as positive controls

  • Include LDHA inhibitors (sodium oxamate) to assess lactate production pathways

• Key findings from recent studies:

  • CCL4 expression is negatively regulated by glucose metabolism in chicken macrophages

  • CCL4 can inhibit glucose metabolism in immune cells

  • The relationship appears bidirectional, with metabolic state affecting CCL4 expression and CCL4 modulating metabolic pathways

Research has demonstrated that treatment with glycolytic inhibitors significantly alters CCL4, suggesting a regulatory feedback loop between this chemokine and cellular energy metabolism . This relationship may be particularly relevant in contexts of viral infection, where both host and pathogen compete for metabolic resources.

What are the molecular mechanisms through which recombinant pig CCL4 exerts antiviral effects?

The antiviral activities of CCL4 involve complex molecular mechanisms that researchers can investigate using the following approaches:

• Receptor engagement analysis:

  • Expression profiling of CCR5 and other CCL4 receptors on target cells

  • Receptor blocking studies using specific antagonists or antibodies

  • CRISPR/Cas9 knockout of receptor genes to confirm specificity

• Signaling pathway investigation:

  • Phosphorylation analysis of downstream kinases (ERK, p38 MAPK, JAK/STAT)

  • NF-κB activation assessment through reporter assays or nuclear translocation

  • Transcription factor binding studies using ChIP assays

• Metabolic reprogramming assessment:

  • Measurement of key metabolites in glycolysis and TCA cycle

  • Analysis of AMPK and mTOR activation states

  • Evaluation of glucose transporter expression and localization

• Viral life cycle interference mechanisms:

  • Viral entry studies using fluorescently labeled viruses

  • Viral replication assessment through measurement of viral RNA/DNA

  • Viral assembly and release quantification using electron microscopy or virus titration

Research has demonstrated that CCL4 can significantly inhibit viral replication in certain models, with experimental evidence showing decreased viral gene expression following CCL4 treatment . The connection between CCL4's metabolic effects and its antiviral activities suggests that metabolic reprogramming may be one mechanism through which this chemokine exerts its protective functions during infection.

How can comparative studies between recombinant pig CCL4 and CCL4 from other species inform structure-function relationships?

Cross-species comparative analyses of CCL4 provide valuable insights into conserved functional domains and species-specific adaptations. Researchers pursuing this approach should consider:

• Sequence and structural comparison methodology:

  • Multiple sequence alignment of CCL4 proteins across species (pig, human, mouse, etc.)

  • Homology modeling based on available crystal structures

  • Molecular dynamics simulations to assess conformational differences

  • Conservation analysis of receptor-binding residues

• Functional comparison experimental design:

  • Parallel chemotaxis assays using identical target cells

  • Receptor binding studies with standardized receptor preparations

  • Cross-species receptor activation using BRET or FRET techniques

  • Calcium flux measurements in response to different CCL4 orthologs

• Relevant findings:

  • Pig CCL4 shares significant sequence homology with human CCL4 but contains unique regions that may alter receptor specificity

  • The core structural elements (beta sheets and disulfide bonds) remain highly conserved across species

  • Species-specific differences in glycosylation patterns may impact stability and activity

• Applications:

  • Rational design of CCL4 variants with enhanced stability or activity

  • Development of species-specific antagonists for research purposes

  • Identification of functionally critical residues through mutational analysis

This comparative approach helps identify which structural features are essential for conserved CCL4 functions across species and which elements contribute to species-specific activities, informing both basic understanding and applied research using recombinant variants.

What are common pitfalls when working with recombinant pig CCL4 and how can they be addressed?

Researchers working with recombinant pig CCL4 frequently encounter several technical challenges. Here are evidence-based solutions to these common issues:

• Protein aggregation and loss of activity:

  • Problem: Recombinant CCL4 can form aggregates during storage or experimental manipulation

  • Solution: Add 0.1-0.5% carrier protein (BSA) to working solutions, avoid excessive freeze-thaw cycles, and maintain appropriate pH (7.2-7.4)

• Endotoxin contamination:

  • Problem: E. coli-derived recombinant proteins may contain endotoxins that confound immunological assays

  • Solution: Use endotoxin-tested preparations or employ additional purification steps (endotoxin removal columns); include polymyxin B controls in sensitive assays

• Tag interference with functional activity:

  • Problem: His-tags or other fusion tags may interfere with CCL4 receptor binding

  • Solution: Compare tagged and untagged versions when possible; use enzymatic tag removal when necessary; include tags in control proteins

• Batch-to-batch variability:

  • Problem: Different production lots may show varying levels of biological activity

  • Solution: Perform activity normalization using standardized bioassays; maintain internal reference standards; include positive controls from previous successful experiments

• Species-specific receptor interactions:

  • Problem: Pig CCL4 may have different receptor affinities compared to human or mouse orthologs

  • Solution: Use species-matched cell systems when possible; validate cross-species activity before using in heterologous systems

Each of these technical considerations should be addressed through careful experimental design and appropriate controls to ensure reliable and reproducible results when working with recombinant pig CCL4.

How can researchers validate the biological activity of recombinant pig CCL4 preparations?

Robust validation of recombinant pig CCL4 biological activity requires multi-parameter assessment using the following methodological approaches:

• Chemotaxis assays:

  • Transwell migration assays using primary porcine monocytes or macrophages

  • Checkerboard analysis to distinguish chemotaxis from chemokinesis

  • Positive controls using established chemoattractants (e.g., CCL2)

  • Dose-response curves to determine optimal concentration range (typically 10-500 ng/mL)

• Receptor binding assays:

  • Radioligand binding using 125I-labeled CCL4

  • Flow cytometry with fluorescently labeled CCL4

  • Surface plasmon resonance for detailed binding kinetics

  • Competitive binding assays with known CCR5 ligands

• Downstream signaling verification:

  • Calcium flux measurement using fluorescent indicators

  • Phosphorylation of ERK1/2 or other MAPK pathway components

  • β-arrestin recruitment assays for receptor activation

  • cAMP modulation assessment when applicable

• Functional cellular responses:

  • T-cell expansion measurement as described in previous sections

  • Cytokine/chemokine induction profiles in target cells

  • Impact on glucose metabolism parameters

  • Antiviral activity in relevant infection models

Researchers should establish validation criteria before beginning experiments, with typical acceptance standards including at least 70-80% activity compared to a reference standard and demonstration of dose-dependent responses across at least three concentration points.

What emerging applications of recombinant pig CCL4 show promise for translational research?

Several innovative applications of recombinant pig CCL4 demonstrate significant translational potential:

• Vaccine adjuvant development:

  • Recombinant CCL4 could enhance vaccine-induced immune responses

  • Preliminary studies show CCL4 can promote T-cell expansion and activation

  • Research design should include dose-optimization studies and comparison with established adjuvants

  • Measurement of both humoral (antibody) and cell-mediated (T-cell) responses is essential

• Metabolic immunotherapy approaches:

  • CCL4's ability to modulate glucose metabolism in immune cells offers therapeutic possibilities

  • Experimental designs should investigate whether CCL4 treatment can reprogram exhausted immune cells in chronic infection models

  • Combination with metabolic modulators may yield synergistic effects

• Antiviral therapeutic strategies:

  • Research suggests CCL4 possesses antiviral properties that could be exploited therapeutically

  • Studies should assess timing of administration (prophylactic vs. therapeutic)

  • Development of stabilized CCL4 variants with enhanced half-life may improve therapeutic potential

  • In vivo models are needed to validate in vitro findings

• Biomarker development:

  • Changes in CCL4 expression during infection may serve as diagnostic or prognostic indicators

  • Longitudinal studies correlating CCL4 levels with disease outcomes are needed

  • Species-specific assays with high sensitivity should be developed

Each of these applications requires rigorous validation in relevant animal models before clinical translation, with particular attention to dose-response relationships, timing of intervention, and potential side effects.

How might the integration of -omics approaches enhance our understanding of CCL4 biology in pigs?

Multi-omics integration offers powerful approaches for comprehensive characterization of CCL4 biology:

• Transcriptomics applications:

  • RNA-seq of CCL4-treated porcine immune cells can reveal global gene expression changes

  • Single-cell RNA-seq enables identification of cell-specific responses to CCL4

  • Time-course analyses can map temporal dynamics of transcriptional responses

  • Analysis should focus on pathway enrichment and transcription factor networks

• Proteomics approaches:

  • Mass spectrometry-based quantitative proteomics of CCL4-stimulated cells

  • Phosphoproteomics to map signaling pathway activation

  • Secretome analysis to identify secondary mediators induced by CCL4

  • Integration with transcriptomics data to identify post-transcriptional regulation

• Metabolomics strategies:

  • Targeted and untargeted metabolomics to quantify changes in metabolic pathways

  • Flux analysis using isotope-labeled metabolites to track metabolic reprogramming

  • Focus on glycolysis, TCA cycle, and fatty acid metabolism intermediates

  • Correlation with CCL4's known effects on glucose metabolism

• Systems biology integration:

  • Network analysis to identify hub genes/proteins in CCL4 response networks

  • Machine learning approaches to predict novel CCL4 functions

  • Mathematical modeling of CCL4 signaling dynamics

  • Cross-species comparative analyses to identify conserved vs. species-specific networks

These multi-omics approaches provide powerful tools for hypothesis generation and mechanistic insights into CCL4 biology beyond traditional reductionist approaches, potentially revealing unexpected functions and therapeutic targets.

What are the key considerations for researchers planning experiments with recombinant pig CCL4?

When designing experiments with recombinant pig CCL4, researchers should prioritize the following evidence-based considerations:

• Protein quality assessment:

  • Verify purity (>95% by SDS-PAGE) and biological activity before experimental use

  • Consider the impact of tags (His, GST) on functional readouts

  • Test for endotoxin contamination, particularly in E. coli-derived preparations

• Experimental design optimization:

  • Include comprehensive controls as outlined in section 4.1

  • Perform dose-response studies (typically 10-500 ng/mL) for each application

  • Consider time-course analyses to capture both immediate and delayed responses

  • Select appropriate readouts based on the specific research question

• Biological context:

  • Use species-matched systems when possible (porcine cells for pig CCL4)

  • Consider the metabolic state of target cells given CCL4's relationship with glucose metabolism

  • Account for potential differences between in vitro and in vivo responses

• Interpretation frameworks:

  • Distinguish between direct CCL4 effects and secondary responses

  • Consider CCL4's dual roles in inflammation and metabolism

  • Integrate findings with existing literature on CCL4 biology across species