GM CSF Human, Sf9

What is the molecular structure of GM-CSF Human produced in Sf9 cells?

GM-CSF Human produced in Sf9 insect cells is a glycosylated, single polypeptide chain containing 127 amino acids with a molecular mass ranging from 14.5-16.5 kDa depending on glycosylation status. The protein exists as a homodimer in its biologically active form . Western blot analysis reveals three distinct glycoforms with molecular masses of 14.5, 15.5, and 16.5 kDa . Following N-glycanase treatment, these multiple bands converge to a single 14.5-15.5 kDa band, confirming that the size heterogeneity stems from differential glycosylation patterns . The signal sequence is properly recognized and cleaved by Sf9 cells, allowing secretion of the mature protein into the culture medium .

How does GM-CSF Human from Sf9 cells differ functionally from E. coli-produced variants?

Unlike bacterially-produced GM-CSF which lacks glycosylation entirely, Sf9-produced GM-CSF exhibits insect-type glycosylation patterns characterized by paucimannose N-glycans. This difference has important functional implications:

All three glycoforms of Sf9-produced GM-CSF demonstrate biological activity in functional assays , making it suitable for most research applications requiring properly folded, active protein.

What are the key biological activities of GM-CSF Human for research applications?

GM-CSF Human expressed in Sf9 cells exhibits several critical biological activities that make it valuable for research:

• Stimulation of growth and differentiation of hematopoietic precursor cells, including granulocytes, macrophages, eosinophils, and erythrocytes

• Enhancement of antigen presentation by dendritic cells and macrophages

• Modulation of macrophage polarization toward either tumor-suppressive M1 or tumor-promoting M2 phenotypes depending on the microenvironment

• Alteration of Th1/Th2 cytokine balance through effects on dendritic cell function

• Inhibition of proliferation in tumor cells expressing GM-CSFR through G0/G1 cell cycle arrest and promotion of differentiation

• Eradication of cancer stem cells through differentiation-promoting effects

These activities make GM-CSF a valuable tool for immunology, cancer research, and hematology studies.

What are the optimal storage conditions for maintaining GM-CSF Human, Sf9 activity?

For optimal stability and activity retention of GM-CSF Human produced in Sf9 cells:

• Add carrier protein (0.1% HSA or BSA) for long-term storage to prevent protein adsorption to container surfaces

• Avoid repeated freeze-thaw cycles as they significantly reduce biological activity

• Store in buffers containing stabilizers such as 10% glycerol (similar to formulations used for related proteins)

• Maintain at -80°C for long-term storage, with working aliquots at -20°C

• Monitor activity periodically through functional assays such as TF1 cell proliferation

Researchers should validate stability under their specific storage conditions through appropriate bioassays to ensure consistent experimental results.

How can researchers optimize baculovirus expression systems for higher yield and quality of GM-CSF Human?

Optimizing GM-CSF production in Sf9 cells requires attention to several parameters:

• Vector design optimization:

  • Employ strong viral promoters like polyhedrin promoter

  • Incorporate efficient signal sequences for secretion (the mouse GM-CSF signal sequence has been successfully used with rat GM-CSF fusion proteins)

  • Consider codon optimization for insect cell expression

• Infection parameters:

  • Determine optimal multiplicity of infection (MOI)

  • Identify peak harvest time post-infection

  • Optimize cell density at infection time

• Post-translational modification control:

  • When studying glycosylation impact, consider tunicamycin treatment which yields exclusively the 14.5 kDa non-glycosylated form

  • For studies requiring homogeneous preparations, implement glycosidase treatments

• Purification strategy:

  • Utilize C-terminal His-tag for affinity chromatography as described in multiple protocols

  • Implement multi-step chromatography for highest purity

Successful production has been achieved through construction of plasmids like pAc373GM-CSF and co-transfection with wild-type baculovirus DNA, with subsequent recombinant virus purification through plaque hybridization .

What challenges exist in developing GM-CSF fusion proteins for targeted immunotherapy?

Developing GM-CSF fusion proteins in Sf9 cells for cancer immunotherapy presents several technical challenges:

• Maintaining dual functionality: Ensuring both GM-CSF domain and targeting/therapeutic domain retain their respective activities. Research examples include GMCSF-NAg fusion proteins where GM-CSF successfully targeted neuroantigen to antigen-presenting cells .

• Glycosylation considerations: Insect cell glycosylation patterns differ from mammalian cells, potentially affecting immunogenicity and pharmacokinetics of therapeutic fusion proteins.

• Balancing dual effects: GM-CSF demonstrates both anti-tumorigenic and pro-tumorigenic effects , requiring careful design to harness beneficial while minimizing detrimental activities.

• Linker design: Selection of appropriate linker sequences between GM-CSF and fusion partners is critical for preserving activity of both domains, ranging from direct fusion to flexible linkers depending on the application .

• Expression and folding: Complex fusion proteins may encounter folding issues, particularly when combining domains with different structural requirements.

Successful approaches include N-terminal GM-CSF fusion with C-terminal therapeutic domains and appropriate linker selection as demonstrated with GMCSF-NAg fusion proteins .

How do purification strategies affect the biological activity of GM-CSF Human?

Purification methodologies significantly impact GM-CSF biological activity:

Critical considerations include:

• Addition of carrier proteins (0.1% HSA or BSA) for stabilization during purification and storage

• Prevention of freeze-thaw cycles to maintain activity

• Removal of endotoxin for applications requiring endotoxin-free material

• Validation of biological activity after each purification step using functional assays

Researchers should optimize purification protocols based on their specific downstream applications, with activity verification at each stage.

What analytical methods are most effective for characterizing GM-CSF Human from Sf9 cells?

Comprehensive characterization of Sf9-produced GM-CSF requires multiple analytical approaches:

• Primary structure verification:

  • SDS-PAGE and Western blotting reveal multiple glycoforms (14.5, 15.5, and 16.5 kDa)

  • Mass spectrometry confirms molecular weight and sequence integrity

  • N-terminal sequencing verifies proper signal peptide cleavage

• Post-translational modifications:

  • Glycosylation analysis through glycosidase treatments (N-glycanase reduces multiple bands to a single form)

  • Treatment with tunicamycin during expression reveals only non-glycosylated (14.5 kDa) species

  • Lectin binding assays characterize glycan types

• Structural integrity assessment:

  • Circular dichroism spectroscopy for secondary structure evaluation

  • Size exclusion chromatography to assess oligomeric state (active form is homodimeric)

  • Differential scanning calorimetry for thermal stability determination

• Functional verification:

  • Cell proliferation assays using GM-CSF-dependent cell lines like TF1

  • Receptor binding assays using recombinant GM-CSF receptor alpha

  • Macrophage or dendritic cell differentiation assays

These complementary methods provide a comprehensive profile of protein quality and functionality.

How can researchers accurately measure GM-CSF potency in functional assays?

Accurately measuring GM-CSF potency requires well-designed functional assays:

• Cell proliferation assays:

  • TF1 erythroleukemic cell proliferation is the gold standard, measuring GM-CSF's ability to stimulate growth in dose-dependent manner

  • Bone marrow colony formation assays provide physiologically relevant measurements

  • Standardization with reference material is essential for relative potency determination

• Differentiation assays:

  • Monitor monocyte-to-macrophage differentiation through morphological changes and marker expression

  • Assess dendritic cell differentiation by measuring surface markers (CD80, CD86, MHC II)

  • Quantify changes in cell surface phenotype by flow cytometry

• Signaling assays:

  • Measure phosphorylation of downstream targets (JAK2/STAT5, ERK, AKT)

  • Utilize reporter gene assays with GM-CSF responsive elements

• Critical controls:

  • Include reference standard GM-CSF in each assay

  • Perform parallel line analysis to determine relative potency

  • Include neutralizing antibodies to confirm specificity

Importantly, all three glycoforms (14.5, 15.5, and 16.5 kDa) of Sf9-produced GM-CSF demonstrate biological activity , so researchers should characterize the glycoform distribution in their preparations.

What experimental designs best elucidate GM-CSF's dual role in tumor microenvironments?

Understanding GM-CSF's "double-edged sword" nature in cancer requires sophisticated experimental approaches:

• In vitro systems:

  • Co-culture tumor cells with immune populations (macrophages, DCs, T cells) with/without GM-CSF

  • Analyze changes in immune cell phenotypes (M1 vs. M2 macrophages, inflammatory vs. tolerogenic DCs)

  • Conduct dose-response and temporal studies to identify concentration and time-dependent effects

• Ex vivo approaches:

  • Culture tumor tissue explants with/without GM-CSF

  • Analyze changes in immune cell composition and activation status

  • Develop patient-derived organoids to test GM-CSF effects in 3D environments

• In vivo experimental designs:

  • Compare tumor models in GM-CSF knockout vs. wild-type mice

  • Create tumor lines with inducible GM-CSF expression

  • Test anti-GM-CSF antibodies or receptor antagonists at different disease stages

• Advanced analysis methods:

  • Single-cell RNA sequencing to profile all cell types in tumor microenvironment

  • Multiplex imaging to spatially resolve GM-CSF effects on different cell populations

  • Systems biology approaches to model complex interactions

The search results highlight GM-CSF's ability to exert both anti-tumorigenic effects (enhancing neutrophil production, M1 macrophage polarization, dendritic cell activation) and pro-tumorigenic effects (converting neutrophils to MDSCs, promoting M2 macrophages, inducing tolerogenic DCs) . These effects vary by cancer type, expression level, and microenvironment context.

How does GM-CSF Human from Sf9 cells contribute to cancer immunotherapy research?

GM-CSF plays multiple roles in cancer immunotherapy research:

• Vaccine adjuvant capability:

  • Enhances antigen presentation by dendritic cells and macrophages

  • Improves anti-tumor effects of melanoma vaccines in combination with checkpoint inhibitors by increasing frequency of antigen-specific, IFN-secreting T cells

  • Alters Th1/Th2 cytokine balance by enhancing antigen-induced immune responses

• Direct anti-tumor effects:

  • Suppresses proliferation of GM-CSFR-expressing tumor cells (colorectal, breast, NSCLC) by inducing G0/G1 cell cycle arrest

  • Eradicates cancer stem cells through promoting differentiation

  • Enhances anti-tumor T cell responses by activating IRF-5 in eosinophils within tumor microenvironment

• Clinical challenges to overcome:

  • GM-CSF can also promote tumorigenesis through MDSC recruitment

  • Varying effects depending on cancer type and microenvironment require careful context-specific application

  • Balancing beneficial vs. detrimental effects requires precise dosing and timing

Researchers using Sf9-produced GM-CSF in cancer models should carefully monitor both pro- and anti-tumor effects to develop optimized therapeutic strategies.

What considerations are important when using GM-CSF in hematological disorder research?

When using GM-CSF Human from Sf9 cells in hematological research:

• Key applications:

  • Studying neutrophil development and function

  • Investigating mechanisms of hematopoietic stem cell mobilization

  • Modeling bone marrow recovery following chemotherapy or radiation

  • Researching GM-CSF receptor alpha (CSF2RA) related disorders

• Methodological considerations:

  • Control glycosylation variation between batches for consistent results

  • Compare effects with E. coli-produced (non-glycosylated) and mammalian-produced variants

  • Include appropriate controls when studying receptor-mediated effects using recombinant GM-CSF receptor alpha

  • Consider physiological concentrations when designing experiments (GM-CSF production is largely activation-dependent)

• Disease-specific applications:

  • Pulmonary surfactant metabolism dysfunction research (related to CSF2RA mutations)

  • Investigating pediatric pulmonary alveolar proteinosis

  • Studying mechanisms of neutropenia and potential therapeutic interventions

Sf9-produced GM-CSF provides a valuable tool for these investigations, balancing proper protein folding with cost-effective production.