GM CSF Human, His
Description
Production and Purification
GM-CSF Human, His is synthesized in Escherichia coli via recombinant DNA technology, followed by proprietary chromatographic purification . Critical steps include:
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Expression: Codon-optimized gene insertion into E. coli vectors.
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Solubility: Requires refolding from inclusion bodies in most cases .
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Formulation: Lyophilized in 10 mM sodium phosphate (pH 7.5) and reconstituted in sterile water .
Biological Activity
The His-tagged variant retains functional equivalence to native GM-CSF in stimulating myeloid progenitor cells:
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Potency: Half-maximal effective concentration (ED₅₀) ≤0.2 ng/mL in TF-1 cell proliferation assays .
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Species Specificity: Active on human, canine, and feline cells but not murine cells .
Mechanistically, GM-CSF binds to a heterodimeric receptor (GM-CSFRα/βc), activating JAK2/STAT5, PI3K/AKT, and MAPK pathways to promote hematopoiesis and immune cell activation .
Research Applications
GM-CSF Human, His is utilized in:
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Immune Cell Differentiation: Induces dendritic cell maturation and macrophage polarization .
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Signal Transduction Studies: Facilitates receptor-ligand interaction assays via His-tag affinity chromatography .
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Therapeutic Development: Serves as a control in immunotherapy trials targeting GM-CSF pathways .
Comparative Analysis with Other GM-CSF Variants
Product Specs
Q&A
Overview of Critical Research Themes
Recent studies have established GM-CSF as a multifunctional cytokine with roles spanning hematopoiesis, immune regulation, and inflammatory pathology. Structural analyses of human GM-CSF complexed with neutralizing antibodies and transgenic models of enhancer function have refined our understanding of its molecular mechanisms, while clinical correlations in autoimmune diseases highlight its therapeutic relevance.
What structural features define recombinant human GM-CSF with a polyhistidine (His) tag, and how do they influence receptor binding?
Recombinant human GM-CSF with a His tag typically retains the native 144-amino acid backbone (14.6 kDa) but incorporates a terminal hexahistidine sequence for affinity purification . X-ray crystallography of GM-CSF bound to neutralizing antibodies (e.g., F1 and 4D4) reveals that epitopes critical for receptor interaction localize to helices A and D . The His tag’s placement at the N- or C-terminus must avoid steric interference with these regions, as shown by comparative binding assays where truncation of the C-terminal helix reduced receptor affinity by 60% . Researchers should validate tag placement via circular dichroism to confirm secondary structure preservation (>90% α-helical content) and surface plasmon resonance to measure binding kinetics to GM-CSFRα (target KD: 1–10 nM) .
How does GM-CSF’s multilineage colony-stimulating activity impact experimental design in hematopoiesis studies?
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Granulocyte-macrophage differentiation: Requires GM-CSF + IL-3 (synergy factor: 3.2×) .
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Erythroid burst formation: Enhanced by GM-CSF + stem cell factor (SCF; 2.5-fold increase in BFU-E colonies) .
Protocols must standardize bone marrow donor age (optimal: 20–35 years) and serum lot (fetal bovine serum <5% variance in colony counts) to minimize variability .
What in vitro models best recapitulate GM-CSF’s role in macrophage polarization?
GM-CSF induces M1-like macrophages with elevated TNF-α (8-fold) and IL-12p70 (12-fold) versus M-CSF-derived M2 phenotypes . Key protocol considerations:
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Cell source: CD14+ monocytes yield 90% CD68+ macrophages after 7 days in 20 ng/ml GM-CSF .
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Activation threshold: LPS priming (10 ng/ml) synergizes with GM-CSF to maximize NF-κB translocation (90% cells at 1 hr) .
Contradictory reports on glycolytic metabolism (e.g., 1.8× vs. 2.5× ATP increase) may stem from oxygen tension differences (5% vs. 21% O₂) .
How do autoantibodies against GM-CSF disrupt signaling, and what experimental strategies mitigate this in pulmonary alveolar proteinosis (PAP) models?
Autoantibodies like F1 bind GM-CSF’s helix A (Kd = 0.8 nM), blocking GM-CSFRα docking . To model PAP:
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Serum transfer: Inject 500 μg F1 IgG into C57BL/6 mice, reducing alveolar macrophage counts by 75% at 14 days .
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GM-CSF mutants: E21R/Q27D substitutions evade F1 binding while retaining 89% receptor activation .
Contradictions in surfactant clearance rates (e.g., 50% vs. 70% rescue) may reflect PPARγ haploinsufficiency in certain strains .
What mechanisms explain GM-CSF’s dual role in neuroinflammation promotion and neuronal protection?
In multiple sclerosis models, GM-CSF:
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Promotes monocyte migration: Increases CCR2 expression 4-fold, enabling blood-brain barrier transmigration (2.3× baseline) .
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Neuronal effects: Binds GM-CSFRβ on astrocytes, upregulating BDNF (1.5×) and reducing glutamate toxicity (IC₅₀ = 15 ng/ml) .
Dose-dependent outcomes occur: ≤10 ng/ml enhances remyelination (30% vs. sham), while ≥50 ng/ml amplifies Th17 infiltration .
How do enhancer polymorphisms affect GM-CSF expression variability in transgenic systems?
The 10.5-kb human GM-CSF transgene (with enhancer) shows copy number-dependent expression (R² = 0.94), whereas enhancer-deleted constructs exhibit 71% variance . Investigators should:
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Map integration sites: Use inverse PCR to exclude position effects.
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Control for CpG methylation: Bisulfite sequencing of the enhancer’s NF-κB site (chr5:132,864,752) identifies hypermethylation-linked silencing .
Data Contradiction Analysis
What metrics validate recombinant GM-CSF protein activity across batches?
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TF-1 proliferation assay: EC₅₀ should be 52.5 ± 8 ng/ml (n = 6 replicates) .
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Receptor phosphorylation: Immunoblot for STAT5 (Y694) after 15-min stimulation (≥3-fold increase over baseline) .
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Endotoxin levels: <0.1 EU/μg via Limulus amebocyte lysate assay .
How can researchers resolve discrepancies in GM-CSF’s reported role in dendritic cell (DC) maturation?
Conflicting DC maturation rates (40% vs. 70% CD83+ cells) arise from IL-4 lot variability . Optimize:
Product Science Overview
Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine that plays a crucial role in the stimulation of proliferation, differentiation, and survival of various hematopoietic cells, including mature neutrophils, macrophages, and dendritic cells . The recombinant form of GM-CSF, particularly the human recombinant version with a His tag, is widely used in research and therapeutic applications.
GM-CSF is a glycoprotein composed of 127 amino acids. The human recombinant version is often tagged with a polyhistidine (His) tag to facilitate purification and detection. This His tag is a sequence of histidine residues that binds strongly to nickel ions, allowing for easy isolation of the protein using nickel-affinity chromatography.
GM-CSF is produced by various cell types, including fibroblasts, endothelial cells, and T lymphocytes, in response to microbial products or inflammatory cytokines . It is essential for the innate immune response, as it stimulates the proliferation and differentiation of granulocytes and macrophages, which are critical for fighting infections.
The production of recombinant human GM-CSF typically involves the expression of the protein in Escherichia coli (E. coli) cells. However, this process often results in the formation of inclusion bodies, which are aggregates of misfolded proteins . To obtain bioactive GM-CSF, these inclusion bodies must be solubilized, refolded, and purified. A simplified method for the efficient refolding and purification of recombinant human GM-CSF has been developed, which does not require extensive experience in protein refolding or purification .
Recombinant human GM-CSF is used as a biotherapeutic agent for immunocompromised individuals, such as those undergoing chemotherapy . It helps to reduce the duration and severity of neutropenia, a condition characterized by low levels of neutrophils, which can lead to severe infections. By stimulating the production of granulocytes and macrophages, GM-CSF enhances the body’s ability to fight infections and recover from chemotherapy-induced neutropenia.
