Flt3 Ligand Human, Sf9

What is Flt3 Ligand and what is its molecular structure when expressed in Sf9 cells?

Flt3 Ligand (FLT3LG) is a hematopoietic four helical bundle cytokine encoded by the FLT3LG gene in humans. When produced in Sf9 insect cells, recombinant human Flt3-Ligand is a glycosylated polypeptide chain containing 155 amino acids that migrates at approximately 19 kDa on SDS-PAGE. The protein's N-terminal sequence has been confirmed to begin with Thr-Gln-Asp-Cys-Ser, and it achieves greater than 95% purity when analyzed by RP-HPLC and SDS-PAGE . Structurally, Flt3 Ligand belongs to the same family as Steel factor and Colony Stimulating Factor 1, sharing homologous features that influence their interaction with tyrosine kinase receptors .

How should recombinant Human Flt3 Ligand Sf9 be stored and reconstituted for optimal stability?

For optimal stability, lyophilized Flt3-Ligand should be stored desiccated below -18°C, although it remains stable at room temperature for up to three weeks. Upon reconstitution, the protein should be stored at +4°C if used within 2-7 days. For long-term storage after reconstitution, temperatures below -18°C are recommended. To prevent protein degradation during freeze-thaw cycles, it is advisable to add a carrier protein such as 0.1% HSA or BSA . When reconstituting the lyophilized protein, it should be dissolved in sterile 18MΩ-cm H₂O at a concentration not less than 100 μg/ml, which can then be further diluted to prepare working solutions for experimental procedures .

How is the biological activity of recombinant Human Flt3 Ligand Sf9 measured and what are typical activity levels?

The biological activity of recombinant Human Flt3 Ligand is typically measured through dose-dependent stimulation of human MUTZ-2 cell proliferation. The effective dose that produces 50% of maximum response (ED₅₀) is less than 3.0 ng/ml for properly functioning protein . This proliferation assay serves as the standard method for assessing Flt3 Ligand functionality in research applications. Quality control specifications typically include endotoxin testing, with acceptable levels being less than 0.1 ng/μg (IEU/μg) of recombinant Flt3-Ligand . Researchers should verify these parameters when establishing experimental protocols to ensure consistent biological effects across studies.

What signaling pathways are activated by Flt3 Ligand binding to its receptor?

When Flt3 Ligand binds to the Flt3 receptor, it induces receptor homodimerization followed by conformational changes that activate receptor tyrosine kinase domains through phosphorylation. This activation rapidly triggers downstream signaling cascades while also initiating internalization and degradation of the receptor homodimer . The key pathways activated include:

• Phosphorylation of Src-homology 2 containing proteins (SHC), which interact with Grb2, Gab2, and SHIP

• Activation of the Ras/MEK/Erk pathway

• Activation of the PI3K pathway

• Phosphorylation of SHC1 and AKT1, leading to activation of the downstream effector mTOR

• Activation of RAS signaling and phosphorylation of downstream kinases including MAPK1/ERK2 and MAPK3/ERK1

• Phosphorylation of FES, FER, PTPN6/SHP, PTPN11/SHP-2, PLCG1, and STAT5A/STAT5B

It's important to note that these signaling mechanisms appear to be cell context-dependent, and activation of wild-type FLT3 causes only marginal activation of STAT5A or STAT5B compared to constitutively active mutants .

How does Flt3 Ligand influence hematopoietic lineage commitment decisions?

Flt3 Ligand plays a crucial instructive role in hematopoietic differentiation, particularly in directing progenitors toward lympho-myeloid fates while inhibiting megakaryocyte/erythrocyte lineage commitment. Research evidence suggests that:

• When Flt3 signaling is activated above a certain threshold by Flt3 Ligand, it leads to upregulation of lymphoid and myeloid lineage-associated genes

• This activation "primes" progenitors toward specific cell fates

• As a result of this priming, Flt3 expression is further increased, and cells acquire the Lymphoid-Primed Multipotent Progenitor (LMPP) phenotype

Experimental evidence from knockout mouse models for both Flt3 Ligand and Flt3 has demonstrated reduction in early lymphoid and myeloid progenitor numbers, confirming its important role in these lineages. This effect appears to be due to both proliferative/survival functions and instructive developmental signaling .

What are optimal experimental conditions for using recombinant Human Flt3 Ligand Sf9 in primary cell cultures?

When designing experiments with recombinant Human Flt3 Ligand Sf9, researchers should consider several critical parameters:

• Concentration range: Effective concentrations typically range from 5-50 ng/ml, with ED₅₀ values less than 3.0 ng/ml in MUTZ-2 cell proliferation assays. Dose-response experiments should be conducted for each specific cell type and application.

• Combination with other cytokines: Flt3 Ligand works synergistically with other growth factors and has limited proliferative activity on its own. Consider combining with appropriate colony stimulating factors and interleukins based on the target cell population .

• Target cell populations: The most responsive populations express the Flt3 receptor, primarily found on primitive hematopoietic progenitor cells and dendritic cells. Receptor expression is restricted to early progenitors with some degree of stem cell activity .

• Culture duration: Optimal exposure times may vary by application, but rapid receptor internalization and degradation following activation suggest that periodic replenishment of Flt3 Ligand may be necessary in long-term cultures.

• Vehicle solution: Following reconstitution in sterile water (at ≥100 μg/ml), further dilutions should be made in appropriate buffers containing carrier protein to prevent adhesion to plastic surfaces and maintain stability .

How can Flt3 Ligand levels be used as a biomarker in hematological research?

Flt3 Ligand serum levels serve as a valuable biomarker of progenitor cell mass and hematopoietic recovery in various research and clinical settings. Key applications include:

For accurate quantification, enzyme-linked immunosorbent assays (ELISAs) are typically employed, with normal serum levels varying by context and requiring appropriate control groups for each experimental setting.

How do mutations in Flt3 receptor affect ligand binding and downstream signaling?

Mutations in the Flt3 receptor can significantly alter its interaction with Flt3 Ligand and subsequent signaling cascades. Research has revealed that:

• Constitutively active Flt3 mutations promote cell proliferation and resistance to apoptosis through aberrant activation of multiple signaling pathways .

• Unlike wild-type receptors that cause only marginal activation of STAT5A/B, mutated Flt3 receptors can drive strong STAT5 signaling, creating distinct cellular responses .

• The inverse relationship between Flt3 expression on leukemic blasts and circulating Flt3 Ligand levels suggests complex regulatory mechanisms that may be exploited in experimental therapeutic approaches .

• Elevated Flt3 Ligand has been observed as a potential mechanism of resistance to Flt3 inhibitor therapies, highlighting the importance of understanding receptor-ligand dynamics when designing targeted interventions .

When studying Flt3 Ligand-receptor interactions, researchers should consider these mutation-dependent effects and incorporate appropriate controls and analytical methods to distinguish physiological from pathological signaling patterns.

What are the technical challenges in comparing Sf9-derived Flt3 Ligand with other expression systems?

Several important technical considerations arise when comparing Flt3 Ligand produced in Sf9 insect cells with other expression systems:

• Glycosylation patterns: Insect cells produce proteins with simpler glycosylation patterns compared to mammalian cells, potentially affecting protein half-life, receptor binding kinetics, and immunogenicity in certain experimental models.

• Activity standardization: Biological activity testing is essential when comparing preparations from different expression systems. The standard MUTZ-2 cell proliferation assay with ED₅₀ < 3.0 ng/ml provides a reference point, but system-specific variations may still exist .

• Structural validation: Analytical techniques including RP-HPLC and SDS-PAGE are necessary to confirm proper folding and structural integrity, particularly important when comparing functional attributes across expression systems .

• Endotoxin contamination: Sf9-derived proteins typically have low endotoxin levels (<0.1 ng/μg), but this should be verified and considered when interpreting experimental results, especially in immunological studies .

• Stability differences: Storage recommendations may vary between expression systems, necessitating optimization of handling protocols for each preparation to maintain consistency in experimental applications.

What evolutionary insights can be gained from studying Flt3 Ligand across species?

Evolutionary analysis of Flt3 Ligand provides valuable insights into its conserved functions and species-specific adaptations:

• Flt3 and its ligand have undergone duplication events during evolution, potentially leading to sub-functionalization in certain species .

• Human Flt3 Ligand can bind to murine Flt3 and activate its downstream signaling, demonstrating significant cross-species conservation of functional domains .

• Flt3 Ligand belongs to a family that includes Steel factor and Colony Stimulating Factor 1, with all three proteins functioning by connecting to and activating unique tyrosine kinase receptors, suggesting evolutionary relationships in hematopoietic regulation .

• Species comparisons have revealed both conserved and divergent roles of Flt3 Ligand in hematopoiesis and dendritic cell development, providing natural experiments that inform our understanding of signaling specificity and redundancy .

Researchers investigating Flt3 Ligand evolution should employ comparative genomics and functional assays across species to decipher the molecular basis for conserved versus species-specific attributes of this important regulatory cytokine.

What are common technical issues when working with recombinant Human Flt3 Ligand Sf9 and how can they be resolved?

When working with recombinant Human Flt3 Ligand Sf9, researchers may encounter several technical challenges:

• Protein aggregation: Upon reconstitution, Flt3 Ligand may form aggregates, reducing activity. Solution: Reconstitute in sterile water at concentrations ≥100 μg/ml and add carrier protein (0.1% HSA or BSA) for further dilutions. Avoid vigorous shaking or vortexing .

• Activity loss during storage: Repeated freeze-thaw cycles significantly reduce biological activity. Solution: Aliquot reconstituted protein before freezing and store with carrier protein for protection .

• Inconsistent cellular responses: Variable responses across experiments may occur due to receptor expression differences or ligand degradation. Solution: Verify Flt3 receptor expression on target cells, use freshly prepared ligand, and include positive control cell lines like MUTZ-2 in parallel assays.

• Interference from endogenous Flt3 Ligand: In primary cell cultures or complex systems, endogenous Flt3 Ligand may confound results. Solution: Consider using Flt3 receptor blocking antibodies as controls to distinguish specific from non-specific effects.

• Solubility issues: Some preparations may show limited solubility in certain buffers. Solution: Reconstitute as recommended in sterile water first, then dilute into final experimental buffer systems .

Careful attention to these technical aspects will enhance reproducibility and reliability of experiments using recombinant Human Flt3 Ligand Sf9.

How can researchers verify the functional activity of Flt3 Ligand in their experimental systems?

Verifying the functional activity of Flt3 Ligand is crucial for experimental validity. Several complementary approaches are recommended:

• Proliferation assays: The standard method uses MUTZ-2 cells with an expected ED₅₀ < 3.0 ng/ml. A dose-response curve should be generated to confirm activity within the expected range .

• Receptor phosphorylation: Western blotting for phosphorylated Flt3 receptor can directly demonstrate ligand-induced activation. Key phosphorylation sites include tyrosine residues in the kinase domain.

• Downstream signaling activation: Measuring phosphorylation of known downstream targets (e.g., ERK1/2, AKT, STAT5) provides functional validation of signal transduction .

• Cell differentiation assays: In hematopoietic progenitor cultures, Flt3 Ligand should promote lympho-myeloid differentiation which can be monitored by flow cytometry for lineage-specific markers .

• Competitive binding assays: Using labeled Flt3 Ligand and displacement with unlabeled protein can verify binding specificity and approximate binding affinity.

Each of these approaches offers complementary information, and combining multiple methods provides the most robust validation of Flt3 Ligand functionality in specific experimental contexts.