MCP1 Human, HEK
Description
Overview of MCP1 and HEK Expression System
Monocyte Chemoattractant Protein-1 (MCP1), also known as C-C motif chemokine ligand 2 (CCL2), is a 76–82 amino acid glycoprotein critical for monocyte/macrophage recruitment during inflammation . In research, recombinant human MCP1 (rhMCP1) is frequently produced in HEK293 cells (human embryonic kidney cells) due to their high protein expression capacity and ability to perform post-translational modifications (e.g., glycosylation) . HEK293-expressed MCP1 retains structural and functional fidelity, making it ideal for studying chemotaxis, signaling pathways, and therapeutic targeting .
Primary Structure
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Amino Acid Sequence: Human MCP1 consists of 76–82 amino acids (depending on expression system), with the core sequence spanning residues 24–99 .
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Glycosylation: HEK293-produced MCP1 is glycosylated, increasing its molecular weight (MW) to 9.5–15 kDa compared to non-glycosylated forms (e.g., 10.9 kDa in E. coli) . Glycosylation slightly reduces chemotactic potency but enhances solubility .
Key Features
| Parameter | HEK293-Produced MCP1 | E. coli-Produced MCP1 |
|---|---|---|
| Glycosylation | Yes | No |
| MW Range | 9.5–15 kDa | ~10.9 kDa |
| Purity | >95% | >95% |
| Activity (ED50) | ≤30 ng/mL | Varies (non-glycosylated) |
| Tag | C-terminal His/Avi | N-terminal His |
Chemotaxis and Signaling
MCP1 binds to CCR2 (its primary receptor) to induce monocyte migration, calcium mobilization, and lamellipodia formation via Rac1 activation . HEK293-expressed MCP1 exhibits robust chemotactic activity (ED50 ≤30 ng/mL), confirmed in THP-1 monocytes and microglial cells .
Pathological Implications
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Atherosclerosis: Recruits monocytes to arterial walls, promoting plaque formation .
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Diabetes/Obesity: Drives insulin resistance and adipose tissue inflammation .
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Cardiac Injury: Induces MCPIP (MCP1-induced protein), a transcription factor linked to cardiomyocyte apoptosis in heart failure .
Regulation
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Hypoxia: Upregulates MCP1 in adipocytes and endothelial cells .
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Cytokines: TNF-α and IL-1β enhance MCP1 transcription via κB and Sp1 binding sites .
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Oncostatin M (OSM): Stimulates MCP1 secretion in keratinocytes and fibroblasts .
HEK293 Expression Workflow
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Cloning: MCP1 cDNA is inserted into a plasmid with tags (e.g., His, Avi) for purification and detection .
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Transfection: HEK293T cells are transfected with linearized plasmids, and integration is verified via PCR .
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Purification: Recombinant MCP1 is purified using nickel-chelating chromatography (His-tag) or biotin-avidin affinity (Avi-tag) .
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Validation: Activity is confirmed via chemotaxis assays (e.g., Boyden chambers) .
Quality Control
Research Models
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Inflammation Studies: HEK293-produced MCP1 is used to mimic monocyte recruitment in atherosclerosis and diabetic nephropathy .
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Signaling Pathways: Investigates CCR2-mediated Rac1 activation and ERM protein interactions .
Therapeutic Targeting
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Inhibitors: Small molecules (e.g., NSC23766) block MCP1-induced Rac1 activation, reducing chemotaxis .
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Gene Therapy: Triplex-forming oligonucleotides (TFOs) suppress MCP1 transcription in HEK293 cells, reducing secretion by ~40% .
Glycosylated vs. Non-Glycosylated MCP1
| Feature | Glycosylated (HEK293) | Non-Glycosylated (E. coli) |
|---|---|---|
| MW | 9.5–15 kDa | 10.9 kDa |
| Chemotactic Potency | High (≤30 ng/mL ED50) | Moderate |
| Solubility | Enhanced | Lower |
| Usage | Functional assays | Structural studies |
Product Specs
CCL2, C-C motif chemokine 2, GDCF-2, HSMCR30, JE, HC11, MCAF, MCP-1, MCP1, Scya2, Sigje, SMC-CF, Immediate-early serum-responsive protein JE, Monocyte chemoattractant protein 1, Small-inducible cytokine A2.
HEK293 Cells.
QPDAINAPVT CCYNFTNRKI SVQRLASYRR ITSSKCPKEA VIFKTIVAKE ICADPKQKWV QDSMDHLDKQ TQTPKTHHHH HH
Q&A
What is MCP1 and why is it commonly studied in HEK cells?
MCP1/CCL2 is a chemokine that plays a key role in inflammatory disorders including atherosclerosis and restenosis . HEK 293T cells provide an ideal expression system because they can be easily transfected with MCP1 genes, support stable protein expression, and enable proper post-translational modifications. These cells have been successfully used to express both human and rabbit MCP1 for various research applications, from inflammatory pathway studies to aptamer selection platforms .
What are the standard methods for detecting MCP1 expression in HEK cells?
Multiple complementary approaches are used to verify MCP1 expression:
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RNA level detection: Real-time PCR and Nanostring analysis can quantify MCP1 mRNA expression
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Protein detection in supernatants: Analysis of 24-hour culture supernatants by immunoassays
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Surface protein verification: Flow cytometry using PE-conjugated monoclonal anti-MCP-1 antibodies (for surface-displayed MCP1)
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Genomic integration confirmation: PCR amplification from chromosomal DNA of transfected cells (expected product sizes: 644 bp for human MCP1, 737 bp for rabbit MCP1)
How can I establish stable MCP1 expression in HEK 293T cells?
The process requires careful construct design and selection:
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Design expression vectors containing the MCP1 gene with appropriate regulatory elements
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Linearize the plasmid before transfection to enhance integration efficiency
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Transfect HEK 293T cells using standard protocols and select for stable integrants
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Verify genomic integration by PCR from chromosomal DNA
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Confirm expression at both RNA level (real-time PCR) and protein level (flow cytometry)
What vector design elements are optimal for MCP1 expression in HEK cells?
Based on successful constructs, the following elements are recommended:
| Component | Recommended Feature | Function |
|---|---|---|
| Vector backbone | pcDNA | Mammalian expression |
| Restriction sites | NheI and XhoI | Cloning boundaries |
| Signal sequence | Murine Igκ chain (76 bp) | Secretion |
| Detection tag | HA tag (26 bp) | Protein identification |
| Target sequence | HMCP-1 (225 bp) or RMCP-1 (303 bp) | Functional chemokine |
| Membrane anchor | PDGFR domain (150 bp) | Surface display |
This design enables efficient secretion and/or cell surface display of MCP1 depending on research requirements .
How can I design constructs to display MCP1 on the surface of HEK cells?
Surface display requires specific construct elements:
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Include a secretion signal sequence (murine Ig kappa-chain signal peptide)
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Insert the MCP1 coding sequence (human or rabbit)
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Add a transmembrane anchoring domain (PDGFR intra-membrane domain)
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Consider including detection tags (HA tag) for verification
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After transfection, verify surface expression by flow cytometry using antibodies against MCP1 or the detection tag
The surface-displayed MCP1 can serve as a target for aptamer selection using cell-SELEX methods or for studying receptor interactions .
What controls should I include when studying cytokine-induced MCP1 expression?
Comprehensive controls are essential:
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Untreated cells as negative controls
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Time-course controls (both 6h and 24h timepoints)
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Isotype control antibodies for flow cytometry
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Verification of signaling pathway activation (STAT3, STAT1, STAT6)
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Quality control validation comparing different detection methods (Nanostring vs. qRT-PCR)
What methods are effective for quantifying MCP1 protein secretion from HEK cells?
MCP1 protein secretion is typically measured from 24-hour culture supernatants . While ELISA is the gold standard, researchers should consider:
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Using species-specific antibodies (human vs. rabbit MCP1 detection)
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Including standard curves with recombinant MCP1
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Normalizing to cell number, culture time, or total protein content
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Statistical analysis with appropriate tests (Mann-Whitney U test or unpaired Welch's t-test)
The timing of collection is critical, as significant protein elevation may only be detectable at specific timepoints (e.g., 24h but not 6h post-stimulation) .
Why might there be discrepancies between MCP1 mRNA and protein expression levels?
Several factors contribute to this common experimental observation:
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Temporal dynamics: In OSM stimulation experiments, MCP1 RNA was elevated at 24h corresponding with increased protein levels, but at 6h, mRNA increases did not translate to statistically significant protein elevation
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Post-transcriptional regulation: RNA stability and translation efficiency
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Secretion kinetics: Delay between protein synthesis and detection in supernatants
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Methodological sensitivity differences: RNA detection methods may be more sensitive than protein assays
How do genetic polymorphisms affect MCP1 expression levels?
The polymorphism at position -2518 (G or A) in the MCP1 gene significantly influences expression:
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G allele carriers produce higher MCP1 levels in response to IL-1β compared to A allele homozygotes
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Allele frequency shows ethnic variation: Japanese populations show 65% G allele (high production) vs. 35% A allele
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This genetic variation affects cytokine responsiveness and may contribute to disease susceptibility
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In Kawasaki disease patients, those with the G allele showed higher serum MCP1 levels, though the difference wasn't statistically significant (p=0.24)
Which signaling pathways regulate MCP1 expression in HEK cells?
Multiple interconnected pathways influence MCP1 expression:
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JAK/STAT pathway: OSM stimulation activates STAT3 in HEK cells, correlating with increased MCP1 expression
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Cytokine signaling: IL-1β induces MCP1, with responses influenced by the -2518 G/A polymorphism
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Transcriptional regulation: The MCP1 promoter region contains elements responsive to inflammatory signals
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Kinase cascades: JAK inhibition with baricitinib can block cytokine-induced signaling pathways that regulate MCP1
How does Oncostatin M (OSM) affect MCP1 expression in HEK cells?
OSM treatment demonstrates a specific pattern of MCP1 induction:
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Significantly elevates MCP1/CCL2 protein levels in culture supernatants at 24 hours
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Markedly increases MCP1/CCL2 mRNA expression at 24 hours
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At 6 hours, shows elevated but not statistically significant differences
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Activates STAT3 signaling, which likely mediates MCP1 upregulation
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Also affects other receptor components including IL-13Rα1 and IL-4Rα
What is the relationship between JAK/STAT signaling and MCP1 in inflammatory models?
JAK/STAT signaling represents a critical pathway connecting cytokine stimulation to MCP1 expression:
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Provides a mechanistic link between inflammatory cytokines and chemokine production
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JAK inhibitors like baricitinib can block cytokine-induced signaling cascades
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In disease models, this pathway connects inflammatory stimuli to monocyte recruitment
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The pathway is implicated in COVID-19 pathophysiology, involving JAK/STAT/APOL1 signaling
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Represents a potential therapeutic target for modulating inflammatory responses
How can HEK cells expressing MCP1 be used for aptamer selection?
HEK cells can be engineered as biological scaffolds for aptamer development:
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Generate HEK cell lines stably displaying human or rabbit MCP1 on their surface
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Use these cells for a toggle cell-SELEX process combining principles of cell and bead-based selection
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Develop aptamers that recognize MCP1 in its native conformation
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Use these aptamers as research tools or potential therapeutic agents
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The approach is particularly valuable for comparative studies between human and rabbit models of atherosclerosis and restenosis
What is the significance of MCP1 polymorphisms in inflammatory disease models?
MCP1 genetic variations have important disease implications:
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Enhanced MCP1 expression is observed in coronary vessels infiltrated by macrophages in fatal Kawasaki disease
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The -2518 G/A polymorphism influences MCP1 production in response to cytokines
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Higher prevalence of the G allele (associated with increased MCP1 production) in Japanese populations may contribute to ethnic differences in disease susceptibility
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This may partially explain why Kawasaki disease preferentially affects Japanese children
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Understanding these polymorphisms is essential when designing experiments and interpreting results across different populations
How can HEK-expressed MCP1 be used for cross-species comparison studies?
HEK cells offer a standardized platform for comparative studies:
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Both human and rabbit MCP1 can be expressed with identical vector designs
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This enables direct comparison of species-specific differences in structure and function
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Such systems are valuable for developing therapeutics that work across species boundaries
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Toggle cell-SELEX with both human and rabbit MCP1-expressing HEK cells can identify cross-reactive aptamers
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This approach is particularly useful for translational research where animal models precede human applications
Product Science Overview
Monocyte Chemotactic Protein-1 (MCP-1), also known as Monocyte Chemotactic and Activating Factor (MCAF) or C-C motif chemokine ligand 2 (CCL2), is a small cytokine belonging to the CC chemokine family . It plays a crucial role in the immune system by recruiting monocytes, memory T cells, and dendritic cells to sites of inflammation produced by either tissue injury or infection . MCP-1 is predominantly secreted by monocytes, macrophages, and dendritic cells .
CCL2 is a monomeric polypeptide with a molecular weight of approximately 13-15 kDa, depending on levels of glycosylation . It is anchored in the plasma membrane of endothelial cells by glycosaminoglycan side chains of proteoglycans . The primary function of CCL2 is to exhibit chemotactic activity for monocytes and basophils, although it does not attract neutrophils or eosinophils . CCL2 also augments monocyte anti-tumor activity and is essential for the formation of granulomas .
CCL2 signals predominantly through the CCR2 receptor . It is expressed by various cell types and attracts not only monocytes and tissue macrophages but also T cells and NK cells to sites of inflammation . Besides chemotaxis, CCL2 is involved in further pro-inflammatory activities such as cell activation, angiogenesis, and fibrosis .
