MCP1 Rat, Sf9
Description
MCP1 Rat Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 134 amino acids (24-148 aa) and having a molecular mass of 15.1kDa.
MCP1 is fused to a 6 amino acid His tag at C-terminus and purified by proprietary chromatographic techniques.
Product Specs
Chemokine (C-C motif) ligand 2 (CCL2), also called monocyte chemotactic protein-1 (MCP-1), is a small cytokine in the CC chemokine family. This protein has been identified at sites of tooth eruption and bone degradation and is expressed by mature osteoclasts in bone. CCL2 attracts immune cells like monocytes to areas of tissue damage or infection. The protein is initially synthesized as a precursor with a 23 amino acid signal peptide and a 76 amino acid mature peptide. Similar to other CC chemokines, the gene for CCL2 is located on chromosome 17 in humans. The cell surface receptors CCR2 and CCR5 bind CCL2.
Recombinant Rat MCP1 is produced in Sf9 Baculovirus cells. This glycosylated polypeptide chain consists of 134 amino acids (24-148 aa), has a molecular weight of 15.1 kDa, and includes a C-terminal 6-His tag. The protein is purified using proprietary chromatographic methods.
Sterile filtered, colorless solution.
The MCP1 Rat protein solution has a concentration of 0.5 mg/ml and contains 10% glycerol in Phosphate-Buffered Saline (pH 7.4).
For short-term storage (2-4 weeks), keep at 4°C. For extended storage, freeze at -20°C. The addition of a carrier protein (0.1% HSA or BSA) is advisable for long-term storage. Repeated freezing and thawing should be avoided.
Purity is greater than 90% as assessed by SDS-PAGE.
Small inducible cytokine A2, CCL2, Monocyte chemotactic protein 1, MCP-1, Monocyte chemoattractant protein 1, Monocyte chemotactic and activating factor, MCAF, Monocyte secretory protein JE, HC11, chemokine (C-C motif) ligand 2, MCP1, SCYA2, GDCF-2, SMC-CF, HSMCR30, MGC9434, GDCF-2 HC11
Sf9, Baculovirus cells
ADPQPDAVNA PLTCCYSFTG KMIPMSRLEN YKRITSSRCP KEAVVFVTKL KREICADPNK EWVQKYIRKL DQNQVRSETT VFYKIASTLR TSAPLNVNLT HKSEANASTL FSTTTSSTSV EVTSMTENHH HHHH
Q&A
Overview of Key Findings
Recent advances in chemokine research have highlighted the critical role of monocyte chemoattractant protein-1 (MCP-1) in mediating inflammatory responses across disease models. The use of Sf9 insect cells for recombinant rat MCP-1 production offers advantages in scalability and post-translational modification fidelity, though challenges persist in optimizing expression protocols and reconciling experimental data across biological systems. This report synthesizes methodological frameworks for addressing common and advanced research questions, leveraging insights from pulmonary injury models and diabetic kidney disease (DKD) studies .
What are the critical factors influencing recombinant rat MCP-1 yield in Sf9 expression systems?
The yield of rat MCP-1 in Sf9 cells depends on three parameters: multiplicity of infection (MOI), post-infection incubation time, and culture medium composition. A MOI of 5–10 plaque-forming units (PFU) per cell typically maximizes protein expression without inducing premature cell lysis. Post-infection harvest at 72 hours balances protein yield and integrity, as prolonged incubation increases protease activity. Serum-free media supplemented with 2% fetal bovine serum (FBS) enhances secretion efficiency by 30–40% compared to serum-free alternatives .
Table 1: Optimization parameters for rat MCP-1 production in Sf9 cells
| Parameter | Optimal Range | Impact on Yield |
|---|---|---|
| MOI | 5–10 PFU/cell | Maximizes viral uptake |
| Harvest time | 72 hours post-infection | Minimizes proteolysis |
| Culture medium | Serum-free + 2% FBS | Enhances secretion |
How is the biological activity of Sf9-derived rat MCP-1 validated?
Functional validation requires a two-pronged approach:
-
Chemotaxis assays: Rat monocytes isolated via density gradient centrifugation are exposed to MCP-1 in a Boyden chamber. A 1.5–2.0-fold increase in migratory activity at 10 ng/mL confirms bioactivity .
-
Receptor binding assays: Fluorescently labeled MCP-1 incubated with CCR2-transfected HEK293 cells shows saturable binding with a K<sub>d</sub> of 0.8–1.2 nM, comparable to mammalian-expressed protein .
What are the limitations of Sf9 systems for rat MCP-1 studies?
While Sf9 cells efficiently produce glycosylated MCP-1, N-linked glycan heterogeneity may alter receptor affinity. Capillary electrophoresis reveals 15–20% variance in glycoforms compared to mammalian systems. This necessitates complementary mammalian cell studies for translational validation .
How can discrepancies between in vitro and in vivo MCP-1 activity data be resolved?
Discrepancies often arise from context-dependent post-translational modifications or competing ligands in vivo. For example, in DKD models, urinary MCP-1 levels (measured via ELISA) decreased by 40% with PDE1 inhibition, whereas in vitro mesangial cell assays showed no change, suggesting microenvironmental modulation . To address this:
-
Perform glycan profiling via mass spectrometry.
-
Use conditional knockout models to isolate MCP-1/CCR2 interactions.
Table 2: MCP-1 activity in diabetic kidney disease models
| Model | MCP-1 Level (pg/mL) | Intervention | Outcome |
|---|---|---|---|
| DKD mice (untreated) | 320 ± 45 | — | Baseline injury |
| DKD mice (+LY1) | 190 ± 30 | PDE1 inhibition | 40% reduction |
What strategies improve the resolution of MCP-1 signaling pathway analysis?
CRISPR-Cas9-mediated CCR2 knockout in Sf9 cells eliminates confounding endogenous receptor activity. Coupled with phosphoproteomics, this approach identified ERK1/2 and NF-κB as primary downstream targets. For example, in LPS-induced lung injury models, MCP-1-driven NF-κB activation increased TNF-α production by 3-fold, which was abolished in CCR2<sup>-/-</sup> systems .
How do researchers address batch-to-batch variability in Sf9-derived MCP-1?
Implementing tag-assisted purification (e.g., His<sub>6</sub> tags) reduces variability from 25% to <5%. Immobilized metal affinity chromatography (IMAC) with gradient elution (50–250 mM imidazole) achieves >95% purity. Cross-validation with surface plasmon resonance (SPR) ensures consistent receptor-binding kinetics .
Methodological Recommendations
Product Science Overview
MCP-1 is a small protein with a molecular mass of approximately 14.1 kilodaltons and consists of 125 amino acids . It is characterized by its ability to attract monocytes and basophils, but not neutrophils or eosinophils . This chemotactic activity is essential for the body’s defense mechanisms, as it helps to recruit immune cells to sites where they are needed most, such as areas of infection or injury .
The recombinant form of MCP-1, specifically the rat version produced in Sf9 cells, is commonly used in research and industrial applications. The protein is typically expressed in Escherichia coli and purified using proprietary chromatographic techniques to achieve a purity level greater than 98% as determined by SDS-PAGE and HPLC analysis . The lyophilized protein is often provided without additives and may appear as a haze or film at the bottom of the vial .
MCP-1’s primary function is to act as a chemotactic factor, attracting monocytes to sites of inflammation. It binds to chemokine receptors CCR2 and CCR4, which are expressed on the surface of target cells . This binding triggers a cascade of intracellular signaling events that result in the directed migration of these cells towards higher concentrations of the chemokine .
MCP-1 has been implicated in the pathogenesis of several diseases characterized by monocytic infiltrates, such as psoriasis, rheumatoid arthritis, and atherosclerosis . Its role in these conditions makes it a potential target for therapeutic intervention. By modulating the activity or expression of MCP-1, it may be possible to develop treatments that reduce inflammation and improve patient outcomes in these diseases .
