M CSF Human, His
Description
MCSF is fused to a 20 amino acid His Tag at N-terminus and purified by proprietary chromatographic techniques.
Product Specs
Q&A
How does the His tag influence the structural integrity and receptor-binding affinity of recombinant human M-CSF?
The His tag’s impact depends on its placement (N-terminal vs. C-terminal) and linker length. Structural studies using X-ray crystallography (2.5 Å resolution) reveal that substitutions at His-9 and His-15 in the N-terminal domain reduce receptor (CSF-1R) binding affinity by 9,100-fold, while C-terminal tags cause minimal disruption . For example, the analog M-CSFα H9A,H15A showed no structural deformation beyond the substituted residues but exhibited near-complete loss of bioactivity . To mitigate interference:
-
Optimal Tag Placement: Use C-terminal His tags with flexible linkers (e.g., GSG linker) to avoid steric hindrance at the receptor-binding interface .
-
Validation: Compare tagged vs. untagged M-CSF using surface plasmon resonance (SPR) to measure CSF-1R binding kinetics (KD values). Tagged variants should maintain sub-nanomolar affinity, as seen in constructs with KD = 0.2–0.5 nM .
What methodological controls are essential when using His-tagged M-CSF in macrophage differentiation assays?
Three critical controls are required:
-
Untagged M-CSF Control: Confirm that His-tagged and native M-CSF induce equivalent CD14+ monocyte differentiation (e.g., 80–90% CD68+ macrophages after 7 days) .
-
Tag-Specific Artifact Check: Use a scramble-tagged negative control (e.g., His tag fused to inert proteins) to rule out non-specific effects.
-
Bioactivity Calibration: Standardize doses using the M-NFS-60 cell proliferation assay (ED50 < 1 ng/mL for validated lots) .
| Parameter | His-Tagged M-CSF | Native M-CSF | Acceptable Deviation |
|---|---|---|---|
| Proliferation (ED50) | 0.8–1.2 ng/mL | 0.5–1.0 ng/mL | ≤20% |
| Osteoclast Formation | 15–20 TRAP+ cells/well | 18–22 TRAP+ cells/well | ≤15% |
| Cytokine Secretion (IL-1β) | 450–600 pg/mL | 500–650 pg/mL | ≤25% |
How do researchers reconcile contradictory findings on His-tagged M-CSF’s role in pro-inflammatory vs. pro-resolving macrophage phenotypes?
Discrepancies arise from stimulus-specific context. For example:
-
LPS Stimulation: His-tagged M-CSF primes macrophages for pro-inflammatory responses, increasing TNF-α (2.5-fold) and IL-1β (3.1-fold) secretion .
-
Resolvin D1 Co-Treatment: Switches macrophages to a pro-resolving phenotype, elevating IL-10 (4.8-fold) and TGF-β (2.3-fold) without altering viability .
To resolve contradictions: -
Stimulus Timing: Pre-treatment with M-CSF for 48hrs before adding polarizing agents reduces variability .
-
Batch Testing: Screen multiple lots for endotoxin levels (<0.1 EU/µg) using the Limulus amebocyte lysate assay, as endotoxin contamination skews cytokine profiles .
What advanced techniques are used to assess the oligomeric state and stability of His-tagged M-CSF?
Three-tiered characterization is recommended:
-
Size-Exclusion Chromatography (SEC): Verify homodimer formation (72–74 kDa expected vs. 36.8 kDa monomer) .
-
Differential Scanning Calorimetry (DSC): Measure thermal stability (Tm = 58–62°C for properly folded variants) .
-
Native PAGE with Western Blot: Detect non-covalent aggregates, which reduce bioactivity by 40–60% if >10% of total protein .
How does His-tagged M-CSF perform in in vivo models compared to native isoforms?
Key considerations include:
-
Pharmacokinetics: His-tagged M-CSF has a shorter half-life (t1/2 = 4–6 hrs) vs. glycosylated isoforms (t1/2 = 18–24 hrs) due to rapid renal clearance .
-
Dosing Regimens: Administer 10–20 µg/kg twice daily in murine models to maintain serum levels >50 ng/mL, sufficient for osteoclast activation .
-
Immunogenicity: His tags elicit anti-tag antibodies in 15–20% of mice after 14 days, necessitating short-term studies .
What are the best practices for troubleshooting low yield in His-tagged M-CSF purification?
Optimize these parameters:
-
Binding Buffer: Use 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole. Avoid reducing agents (DTT/TCEP) to preserve disulfide bonds .
-
Elution Gradient: Stepwise imidazole elution (50 mM → 250 mM) improves purity from 70% to >95% .
-
Protease Inhibition: Include 1 mM PMSF during lysis to prevent cleavage at Arg257-Pro258, a common degradation site .
How can researchers validate the species specificity of His-tagged human M-CSF in cross-species experiments?
Human M-CSF binds murine CSF-1R with 60–70% efficiency relative to murine M-CSF . Validate using:
Product Science Overview
M-CSF is a homodimeric glycoprotein growth factor that exerts its biological effects by signaling through a receptor tyrosine kinase known as CSF-1R or M-CSF-R, which is encoded by the c-fms proto-oncogene . The stimulation of CSF-1R upon binding to M-CSF activates several signaling pathways, including MAPK, PI3K, and PLCγ . These pathways are essential for the development and function of mononuclear phagocytic cell lineages, including monocytes, macrophages, and osteoclasts .
M-CSF is involved in various physiological processes, such as:
- Regulation of Inflammatory Responses: M-CSF works in conjunction with other stimuli like IFN-γ, LPS, and IL-4 to regulate inflammatory responses .
- Bone Resorption: It is required for bone resorption by osteoclasts .
- Development and Regulation of Tissues: M-CSF plays a role in the development and regulation of the placenta, mammary gland, and brain .
- Immunological Defense: It is crucial for immunological defense mechanisms .
- Fertility and Pregnancy: M-CSF has been shown to play important roles in fertility and pregnancy .
Recombinant human M-CSF is produced using E. coli expression systems and is often tagged with a His tag to facilitate purification . This recombinant protein is used extensively in research to study the differentiation of macrophages from peripheral blood monocytes, the differentiation of osteoclasts from CD14+ monocytes, and in various cell culture and functional assays .
Recombinant M-CSF can be used for a variety of applications, including:
