Recombinant Human C-C motif chemokine 20 protein (CCL20) (Active)

What is Recombinant Human CCL20 and what are its alternative names?

Recombinant Human CCL20 is a small cytokine belonging to the CC chemokine family that is produced through recombinant DNA technology, typically expressed in E. coli systems . The protein is also known by several alternative names including Liver Activation Regulated Chemokine (LARC), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), and Exodus . These naming conventions reflect historical discoveries of the protein in different contexts and by different research groups, but all refer to the same molecular entity encoded by the CCL20 gene .

What is the amino acid sequence and molecular structure of human CCL20?

The mature recombinant human CCL20 consists of 70 amino acids with the sequence: ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM . This sequence corresponds to the Ala27-Met96 portion of the full protein, representing the mature, processed form after signal peptide cleavage . The molecular weight of CCL20 is approximately 8-9.3 kDa, with precise measurements placing it at 8025.5 Daltons . Under certain electrophoretic conditions (Bis-Tris PAGE), the protein may migrate to appear as 12-14 kDa due to its structural properties and interaction with the gel matrix .

What are the primary biological functions of CCL20?

CCL20 functions as a chemotactic factor that strongly attracts lymphocytes and weakly attracts neutrophils . It specifically binds to the G protein-coupled receptor CCR6, inducing chemotactic migration in a wide range of CCR6-expressing cells including immature dendritic cells (DC), effector/memory T-cells, and B-cells . CCL20 plays crucial roles in the formation and function of mucosal lymphoid tissues by facilitating the chemoattraction of lymphocytes and dendritic cells toward epithelial cells surrounding these tissues . Additionally, CCL20 is expressed in the skin and contributes to the chronic inflammation characteristic of psoriasis . The protein also promotes adhesion of memory CD4+ T cells and inhibits colony formation of bone marrow myeloid immature progenitors .

How should recombinant CCL20 be reconstituted and stored for maximum stability?

For optimal reconstitution, lyophilized recombinant CCL20 should be dissolved in sterile water (18 MΩ·cm quality) to a concentration of at least 0.1 mg/ml . This stock solution can be further diluted if required for specific experimental applications. For storage stability, the following guidelines are recommended:

• Store lyophilized protein desiccated below -18°C or preferably at -80°C for up to 12 months

• After reconstitution, store at 4°C for short-term use (up to 7 days)

• For reconstituted long-term storage, maintain at -20°C to -80°C

• For extended storage periods, it is beneficial to add a carrier protein (0.1% HSA or BSA)

• Aliquot the protein into smaller quantities to minimize freeze-thaw cycles, which significantly reduce biological activity

The stability of CCL20 at different storage conditions is summarized in the table below:

What assays can be used to measure CCL20 biological activity in vitro?

The biological activity of recombinant CCL20 can be assessed through several complementary approaches:

• Chemotaxis Assays: The primary functional assay measures the ability of CCL20 to chemoattract human T lymphocytes, typically using a concentration range of 10-50 ng/ml . A specific activity of 20,000-100,000 U/mg is indicative of fully functional protein . This can be quantified using Transwell migration chambers or similar systems.

• Receptor Binding Assays: CCL20 binding to CCR6 receptors can be measured using cells expressing CCR6, with competitive binding assays utilizing labeled CCL20 or displacement studies.

• Calcium Mobilization: As CCL20 binding to CCR6 triggers intracellular calcium release, fluorescent calcium indicators can monitor this response in CCR6-expressing cells.

• Cell Adhesion Assays: Since CCL20 promotes the adhesion of memory CD4+ T cells, adhesion assays can be employed as functional readouts.

• Colony Formation Inhibition: The ability of CCL20 to inhibit colony formation of bone marrow myeloid immature progenitors can serve as a functional assessment .

All recombinant CCL20 preparations should be validated for endotoxin content (<0.01 EU/μg of protein) to ensure that cellular responses are attributable to CCL20 and not to bacterial contaminants .

How do post-translational modifications affect CCL20 function in different expression systems?

While E. coli-expressed recombinant CCL20 is biologically active, it lacks post-translational modifications (PTMs) present in mammalian-expressed variants . Research indicates several important considerations:

• Glycosylation: Mammalian-expressed CCL20 may exhibit N-linked or O-linked glycosylation, potentially affecting receptor binding kinetics and protein stability. E. coli-derived CCL20 lacks these modifications but maintains core biological activity.

• Disulfide Bonds: The correct formation of disulfide bonds is critical for CCL20 tertiary structure and function. While E. coli systems can produce correctly folded protein, specialized strains or oxidizing conditions may be required to ensure proper disulfide formation.

• Functional Differences: Comparative studies between E. coli-derived and mammalian-expressed CCL20 show similar specific activities in chemotaxis assays, but potentially different half-lives in circulation and receptor binding affinities.

When designing experiments, researchers should consider which expression system best serves their research questions. For basic receptor binding and chemotaxis studies, E. coli-derived protein is typically sufficient, while studies examining in vivo pharmacokinetics or complex interactions may benefit from mammalian-expressed variants.

What is known about CCL20's role in neuroinflammatory conditions?

Recent evidence suggests significant involvement of CCL20 in neuroinflammatory conditions, particularly in subarachnoid hemorrhage (SAH) . Key findings include:

• Upregulation Pattern: CCL20 is upregulated in SAH mouse models and in cultured primary microglia and neurons following hemorrhagic injury .

• Therapeutic Potential: Neutralization of CCL20 using specific antibodies has demonstrated neuroprotective effects, including:

  • Alleviation of SAH-induced neurological deficits

  • Reduction in brain water content (edema)

  • Decreased neuronal apoptosis

  • Suppression of microglial activation

• Signaling Mechanism: The CCL20-CCR6 axis appears to mediate inflammatory cascade activation in the central nervous system, potentially through NF-κB and MAPK pathways.

These findings suggest CCL20 as a potential therapeutic target in neuroinflammatory conditions, with implications for stroke, traumatic brain injury, and other acute central nervous system injuries where inflammatory cascades contribute to secondary tissue damage.

What controls should be included when studying CCL20 in chemotaxis assays?

When designing chemotaxis experiments with CCL20, several controls are essential for rigorous interpretation:

• Negative Controls:

  • Buffer-only conditions (vehicle control)

  • Heat-denatured CCL20 (to confirm activity is protein-dependent)

  • Non-chemotactic proteins of similar molecular weight

  • Random migration controls (equal concentration in both chambers)

• Positive Controls:

  • Known chemotactic factors for the cell type being tested (e.g., CXCL12 for lymphocytes)

  • Commercial CCL20 standard with verified activity

• Specificity Controls:

  • CCR6 receptor blockers or neutralizing antibodies

  • CCR6 knockout cells or CCR6 siRNA-treated cells

  • Chemically modified CCL20 with altered receptor binding domains

• Dose-Response Assessment:

  • Multiple concentrations of CCL20 ranging from 1-100 ng/ml

  • Optimal chemotactic response typically occurs at 10-50 ng/ml

A checkerboard analysis (varying CCL20 concentrations in both upper and lower chambers) can help distinguish between true chemotaxis (directional migration) and chemokinesis (increased random motility).

How do different CCL20 formulations affect experimental results?

Different commercial preparations of recombinant CCL20 may contain variations that affect experimental outcomes:

• Fusion Tags: Some recombinant CCL20 products contain N-terminal His-tags or other fusion elements . Research indicates:

  • His-tagged CCL20 maintains biological activity but may show altered receptor binding kinetics

  • Tag-free CCL20 more closely resembles native protein but may have lower solubility

  • Fusion partners may interfere with certain antibody epitopes in immunological assays

• Formulation Buffer Components:

  • PBS-formulated CCL20 (pH 7.4, containing 150 mM NaCl) provides physiological compatibility

  • Carrier proteins may be present in some formulations to enhance stability

  • Preservatives or stabilizers may influence sensitive cell-based assays

• Endotoxin Levels: High-quality preparations contain <0.01 EU/μg of protein . Higher endotoxin levels can:

  • Independently activate immune cells, confounding chemotaxis results

  • Trigger TLR4 signaling, which may synergize with or antagonize CCR6 pathways

  • Lead to false-positive inflammatory responses in cell culture

When comparing results across studies, researchers should account for these formulation differences and ideally validate key findings with preparations from multiple sources.

What are the emerging therapeutic applications of CCL20 inhibition or supplementation?

Recent research has identified several promising therapeutic applications targeting the CCL20-CCR6 axis:

• Neuroinflammatory Conditions:

  • CCL20 neutralization reduces neurological deficits following subarachnoid hemorrhage

  • This approach decreases neuronal apoptosis and edema formation

  • Similar strategies may apply to ischemic stroke and traumatic brain injury models

• Autoimmune Disorders:

  • CCL20 contributes to chronic inflammation in psoriasis

  • Blocking CCL20-CCR6 interactions shows promise in reducing skin inflammation

  • Similar approaches may benefit other autoimmune conditions where CCR6+ cells are implicated

• Mucosal Immunity Enhancement:

  • Controlled CCL20 supplementation may enhance mucosal vaccine responses

  • This approach potentially increases dendritic cell and lymphocyte recruitment to vaccination sites

  • Studies suggest improved antigen presentation and memory T-cell generation

• Cancer Immunotherapy:

  • CCL20 may enhance tumor infiltration by specific lymphocyte subsets

  • Engineered CCL20 gradients could increase immune cell access to poorly infiltrated tumors

  • Combination approaches with checkpoint inhibitors are under investigation

These therapeutic directions highlight the dual nature of CCL20 as both a potential target for inhibition in inflammatory conditions and a possible adjuvant for enhancing desired immune responses.

How do researchers differentiate between direct CCL20 effects and secondary responses?

Distinguishing primary from secondary effects presents a significant challenge in CCL20 research. Methodological approaches include:

• Temporal Analysis:

  • Short-term assays (minutes to hours) more likely reflect direct CCL20 effects

  • Longer observations may capture secondary cytokine/chemokine cascades

  • Time-course experiments with frequent sampling can map response progression

• Pathway Inhibition Studies:

  • Specific CCR6 antagonists block direct CCL20 effects

  • Inhibitors of downstream signaling (PI3K, MAPK, etc.) help delineate mechanism

  • Combined inhibition of multiple pathways can reveal redundancy or synergism

• Transcriptomic/Proteomic Approaches:

  • RNA-seq at multiple time points following CCL20 stimulation reveals response dynamics

  • Proteomics identifies changing cellular protein landscapes

  • Bioinformatic pathway analysis distinguishes primary from secondary response patterns

• Single-Cell Analysis:

  • Flow cytometry with phosphoprotein markers identifies directly responding cells

  • Single-cell RNA-seq distinguishes cell-specific responses

  • Imaging approaches can track cellular responses in real-time

These methodological approaches allow researchers to construct detailed models of CCL20-initiated signaling cascades and their biological consequences.

What are common pitfalls in CCL20 functional assays and how can they be addressed?

Researchers frequently encounter several challenges when working with CCL20:

• Loss of Activity During Storage/Handling:

  • Problem: Repeated freeze-thaw cycles significantly reduce biological activity

  • Solution: Aliquot reconstituted protein into single-use volumes and maintain at recommended temperatures

• Inconsistent Chemotaxis Results:

  • Problem: Variable cell responses to seemingly identical CCL20 concentrations

  • Solutions:

    • Ensure consistent cell culture conditions and passage numbers

    • Verify CCR6 expression levels on target cells before assays

    • Use freshly prepared chemotaxis media without serum (which may contain chemokines)

    • Allow for equilibration of chamber temperature and CO₂ levels before analysis

• Endotoxin Contamination:

  • Problem: Bacterial endotoxins activate immune cells independently of CCL20

  • Solutions:

    • Use validated low-endotoxin preparations (<0.01 EU/μg)

    • Include polymyxin B controls to neutralize potential endotoxin effects

    • Consider endotoxin testing of working solutions if inconsistencies arise

• Protein Adsorption to Plastics:

  • Problem: CCL20 may adhere to plastic surfaces, reducing effective concentration

  • Solutions:

    • Use low-binding tubes and plates

    • Include carrier protein (0.1% BSA) in dilution buffers

    • Pre-coat plastic surfaces with BSA solution

• Receptor Desensitization:

  • Problem: Prolonged exposure to CCL20 causes CCR6 downregulation

  • Solutions:

    • Design time-course experiments to account for desensitization

    • Include receptor recycling inhibitors if mechanisms of desensitization are being studied

    • Use pulse-chase approaches rather than continuous stimulation

Addressing these common issues will significantly improve experimental reproducibility and data interpretation in CCL20 research.

What are the key knowledge gaps in CCL20 biology that require further investigation?

Despite significant advances in understanding CCL20 biology, several important knowledge gaps remain:

• Structure-Function Relationships: While the primary sequence of CCL20 is well-established, detailed structural studies examining how specific domains interact with CCR6 and how post-translational modifications affect these interactions are still needed .

• Cell-Specific Responses: Different CCR6-expressing cell populations may respond differently to CCL20 stimulation. More research is needed to characterize these differential responses and their biological significance .

• In Vivo Regulation: The mechanisms controlling CCL20 expression in different tissues under physiological and pathological conditions remain incompletely understood, particularly regarding the timing and magnitude of expression in response to inflammatory stimuli .

• Therapeutic Targeting: While CCL20 inhibition shows promise in conditions like subarachnoid hemorrhage, optimal delivery methods, dosing regimens, and potential side effects of long-term CCL20-CCR6 axis modulation require further investigation .

• Cross-Talk with Other Chemokine Systems: How the CCL20-CCR6 axis interacts with other chemokine systems in complex immune responses represents an important area for future research, particularly in the context of inflammatory disorders and cancer immunology.

Addressing these knowledge gaps will require multidisciplinary approaches combining structural biology, cell signaling studies, in vivo models, and clinical investigations. Such research will not only enhance our fundamental understanding of CCL20 biology but may also reveal new therapeutic opportunities across multiple disease areas.