FGF23 Human, Sf9

What is the basic structure of human FGF23 protein?

Human FGF23 is a 32-kDa glycoprotein consisting of 251 amino acids with three distinct domains: a 24-amino acid signal sequence, a 155-amino acid N-terminal FGF core homology domain, and a 72-amino acid C-terminal domain unique to FGF23. Between the N- and C-terminal domains lies a proteolytic site (176RXXR179) where FGF23 can be cleaved by subtilisin-like proprotein convertase and plasminogen activators, resulting in two fragments - an inactive N-terminal fragment and a C-terminal fragment that can act as a natural FGF23 antagonist by competitively interfering with the formation of intact FGF23/αKlotho/FGFR complex .

What are the primary physiological functions of FGF23?

FGF23 functions primarily as a phosphaturic hormone that regulates phosphate homeostasis. It acts on the kidney to promote phosphaturia and suppress active vitamin D (1,25-dihydroxyvitamin D3) synthesis, thereby decreasing intestinal phosphate absorption and promoting negative external phosphate balance . FGF23 inhibits the expression of type II Na-dependent phosphate co-transporters (NaPi-2a and NaPi-2c) in the renal proximal tubule . Additionally, FGF23 has been associated with cardiac effects, particularly with heart failure with preserved ejection fraction (HFpEF), as indicated by Mendelian randomization studies .

What are the optimal conditions for expressing biologically active human FGF23 in Sf9 cells?

Based on protein expression principles and FGF23 characteristics, optimal conditions for expressing human FGF23 in Sf9 cells would include:

• Vector design incorporating the full 251 amino acid sequence with an appropriate signal sequence

• MOI (multiplicity of infection) optimization to prevent cellular toxicity while maximizing protein yield

• Temperature control (typically 27-28°C for Sf9 cells)

• Harvest timing optimization (typically 48-72 hours post-infection)

• Addition of protease inhibitors to prevent degradation of the sensitive proteolytic site (176RXXR179)

The FGF23 expressed in insect cells would need to be purified using chromatographic techniques similar to those mentioned for other recombinant FGF23 preparations .

How should researchers design validation experiments to confirm functionality of Sf9-expressed FGF23?

Functional validation of Sf9-expressed FGF23 should include:

• Proliferation assay: Testing the protein's biological activity using NIH/3T3 mouse embryonic fibroblasts. The expected ED50 would be approximately 0.05-0.5μg/ml in the presence of 5μg/ml Recombinant Mouse Klotho and 10 μg/ml of HPR .

• Receptor binding assay: Confirming the formation of the FGFR/FGF23/αKlotho tertiary complex, which is essential for FGF23 signaling .

• Phosphate transport inhibition: Measuring the protein's ability to downregulate NaPi-2a and NaPi-2c in renal proximal tubule cell models .

• Vitamin D suppression assay: Assessing the suppression of 1,25-(OH)2D synthesis in appropriate cell models .

What purification strategies yield the highest purity and recovery of intact FGF23 from Sf9 culture?

For optimal purification of FGF23 from Sf9 culture, researchers should consider:

• Initial clarification: Centrifugation or filtration to remove cellular debris

• Capture step: Affinity chromatography using tagged constructs or specific FGF23 binding partners

• Intermediate purification: Ion exchange chromatography to separate intact FGF23 from proteolytic fragments

• Polishing step: Size exclusion chromatography to ensure homogeneity

• Stabilization: Addition of carrier proteins (0.1% HSA or BSA) for long-term storage to prevent aggregation

• Freeze-thaw prevention: Aliquoting to prevent repeated freeze-thaw cycles that can degrade the protein

How do post-translational modifications of FGF23 in Sf9 affect its interaction with αKlotho and FGFR?

Post-translational modifications, particularly glycosylation patterns, can affect the binding affinity and specificity of FGF23 to the αKlotho/FGFR complex. The current model of this complex is a 2:2:2 ternary structure where αKlotho simultaneously tethers the D3 domain of FGFR1c and FGF23 via the FGF23 C-terminal tail .

Differences in glycosylation between mammalian and Sf9-expressed FGF23 may alter:

• The stability of this complex formation

• Binding kinetics and affinity

• Downstream signal transduction efficiency

Researchers should validate these interactions experimentally when using Sf9-expressed FGF23, especially for structural studies or precise signaling experiments.

What are the key differences in proteolytic processing of FGF23 between Sf9 and mammalian expression systems?

The proteolytic processing at the 176RXXR179 site of FGF23 is a critical regulatory mechanism that may differ between expression systems. In Sf9 cells:

• The subtilisin-like proprotein convertases present in mammalian systems may be absent or functionally different

• The proteolytic cleavage rate may be altered, potentially resulting in higher yields of intact FGF23

• The ratio of intact to cleaved FGF23 may need to be carefully monitored and characterized

These differences could be advantageous for producing stable, intact FGF23 for research purposes, but researchers must validate the proteolytic status of their preparation through methods like Western blotting or mass spectrometry.

How does temperature and pH stability compare between native and Sf9-expressed FGF23?

While specific data on Sf9-expressed FGF23 stability is not provided in the search results, general protein expression principles suggest:

• Temperature stability: Sf9-expressed proteins may show different thermal denaturation profiles due to altered post-translational modifications

• pH stability: The isoelectric point and pH stability range might differ slightly due to modifications

• Storage stability: Addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage regardless of expression system

• Freeze-thaw sensitivity: Prevention of freeze-thaw cycles is critical for maintaining activity

Researchers should empirically determine these parameters for their specific Sf9-expressed FGF23 preparation.

How can Sf9-expressed FGF23 be effectively used in kidney phosphate transport studies?

For kidney phosphate transport studies, Sf9-expressed FGF23 can be utilized to:

• Investigate NaPi-2a and NaPi-2c regulation: FGF23 downregulates these transporters in renal proximal tubules, leading to phosphaturia .

• Design ex vivo experiments: Similar to studies with isolated tubules from Hyp mice with high FGF23 levels, researchers can examine how exogenous FGF23 affects phosphate transport recovery and NaPi-2a trafficking to the apical membrane .

• Study receptor specificity: FGF23 primarily signals through FGFR1 in the kidney, with FGFR4 playing a minor role. Researchers can use Sf9-expressed FGF23 to investigate these receptor interactions in various kidney tubule models .

• Temporal response studies: Track the time course of NaPi-2a (rapid, <1 hour), PiT-2 (>8 hours), and NaPi-2c (>24 hours) downregulation following FGF23 exposure .

What experimental controls are essential when studying FGF23-Klotho interactions using Sf9-derived proteins?

When studying FGF23-Klotho interactions with Sf9-expressed proteins, essential controls include:

• Native FGF23 comparison: Include mammalian-expressed FGF23 as a positive control to benchmark activity

• C-terminal fragment control: Include the C-terminal fragment of FGF23 as a natural antagonist control

• Klotho dependency: Compare FGF23 activity in the presence and absence of αKlotho to confirm co-receptor dependency

• FGFR isoform specificity: Test multiple FGFR isoforms to confirm the expected receptor specificity pattern

• Signal transduction validation: Confirm activation of downstream pathways using phosphorylation-specific antibodies

These controls help ensure that Sf9-expressed FGF23 maintains the expected biological interactions with its co-receptor and signaling partners.

How can researchers accurately measure the biological activity of Sf9-expressed FGF23 in comparison to mammalian-expressed standards?

To accurately measure biological activity of Sf9-expressed FGF23:

• Cell proliferation assay: The standard bioassay uses NIH/3T3 mouse embryonic fibroblasts with an expected ED50 of 0.05-0.5μg/ml in the presence of 5μg/ml Recombinant Mouse Klotho and 10 μg/ml of HPR .

• Phosphate transport inhibition: Measure the reduction in sodium-dependent phosphate transport in proximal tubule cell models or brush border membrane vesicles.

• Dose-response curves: Generate full dose-response curves with both Sf9 and mammalian-expressed FGF23 to compare potency and efficacy.

• Relative potency calculation: Calculate the relative potency using parallel line bioassay methodology to provide a quantitative comparison of activity.

• Western blot of downstream signaling: Measure phosphorylation of downstream signaling molecules at equivalent concentrations of both proteins.

How can Sf9-expressed FGF23 be utilized in cardiac research models examining heart failure pathophysiology?

Given the association between FGF23 and heart failure, particularly HFpEF , Sf9-expressed FGF23 could be utilized in cardiac research through:

• Cardiomyocyte hypertrophy models: Treating isolated cardiomyocytes with FGF23 to study the induction of prohypertrophic genes and hypertrophic responses.

• Ex vivo cardiac function studies: Perfusing isolated hearts with FGF23 to examine acute effects on contractility, relaxation, and electrophysiology.

• Co-culture systems: Establishing co-cultures of cardiomyocytes and kidney cells to study organ crosstalk mediated by FGF23.

• Receptor selectivity studies: Using FGF23 alongside specific FGFR inhibitors to determine which receptors mediate cardiac effects.

• Mechanistic investigations: Examining whether cardiac effects depend on αKlotho co-receptor or if there are direct, Klotho-independent cardiac actions of FGF23.

These approaches could help clarify the relationship between FGF23 and heart failure with preserved ejection fraction observed in Mendelian randomization studies .

What are the critical considerations when designing FGF23 antagonist screening assays using Sf9-expressed protein?

For FGF23 antagonist screening using Sf9-expressed FGF23:

• Assay format selection: Choose between cell-based (signaling) or biochemical (binding) assays

• Control antagonist: Include the C-terminal fragment of FGF23 as a natural antagonist positive control

• Signal window optimization: Ensure adequate dynamic range by optimizing FGF23 and αKlotho concentrations

• Counter-screening: Develop parallel assays to identify non-specific inhibitors

• Validation strategy: Establish secondary and tertiary assays to confirm hits from primary screens

• Characterization of inhibition mechanisms: Differentiate between compounds that:

  • Directly bind FGF23

  • Interfere with FGF23-αKlotho interaction

  • Block FGF23-FGFR binding

  • Inhibit formation of the ternary complex

How does the FGF23-Klotho-FGFR signaling complex formation differ between soluble and membrane-bound forms of αKlotho?

The search results indicate important differences in complex formation between soluble and membrane-bound αKlotho:

• Traditional model: Membrane αKlotho functions as a mandatory co-receptor for FGF23 along with FGFR to transduce FGF23 signaling .

• Alternative model: Soluble αKlotho can also sustain FGF23 signaling by acting as a "deliverable" co-receptor .

• Complex structure: The current model based on crystal structure is a 2:2:2 ternary complex of αKlotho, FGFR1c, and FGF23, where αKlotho simultaneously tethers the D3 domain of FGFR1c and FGF23 via the FGF23 C-terminal tail .

• Binding mechanism: In this complex, αKlotho implements FGF23-FGFR1c proximity and confers stability, potentially with different kinetics or stability when using soluble versus membrane-bound forms .

• Experimental validation: In vitro experiments show that supraphysiologic concentrations of soluble αKlotho protein can form a similar complex as membrane αKlotho to transduce FGF23 signal, but in vivo confirmation is lacking .

Understanding these differences is crucial when designing experiments using Sf9-expressed FGF23 in combination with different forms of αKlotho.

What are common pitfalls in interpreting FGF23 activity data and how can they be avoided?

Common pitfalls in FGF23 activity data interpretation include:

• Proteolytic degradation misinterpretation: Reduced activity may be due to proteolytic cleavage at the 176RXXR179 site rather than inherent low activity. Use Western blotting to confirm intact protein before activity testing .

• Co-receptor dependency overlooked: FGF23 requires αKlotho to activate FGFRs efficiently. Ensure adequate αKlotho is present in activity assays .

• FGFR isoform specificity: Different tissues express different FGFR isoforms with varying affinity for the FGF23/αKlotho complex. Confirm appropriate receptor expression in your experimental system .

• Improper controls: Always include positive controls (native FGF23) and negative controls (heat-inactivated FGF23) in activity assays.

• Storage-related activity loss: Activity loss may occur due to improper storage. Addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage, and freeze-thaw cycles should be prevented .

How should researchers address contradictory results between different FGF23 functional assays?

When faced with contradictory results between different FGF23 functional assays:

• Verify protein integrity: Confirm that the FGF23 preparation is intact and not degraded using Western blot analysis.

• Check co-receptor and receptor expression: Verify that test systems express appropriate levels of αKlotho and FGFR isoforms necessary for FGF23 signaling.

• Consider assay sensitivity differences: Different assays may have varying sensitivity to detect FGF23 activity. Cell proliferation assays typically have an ED50 of 0.05-0.5μg/ml in the presence of appropriate co-factors .

• Evaluate time-dependent responses: Some FGF23 effects are rapid (e.g., NaPi-2a downregulation < 1 hour) while others are delayed (NaPi-2c > 24 hours) .

• Assess indirect vs. direct effects: FGF23 can have both direct effects on target cells and indirect effects through modulation of other hormones like PTH and 1,25(OH)2D.

• Perform dose-response studies: Generate complete dose-response curves to identify potential biphasic responses or ceiling effects.

What quality control parameters should be monitored during large-scale production of FGF23 in Sf9 cells?

For large-scale production of FGF23 in Sf9 cells, critical quality control parameters include:

• Protein integrity: Regular SDS-PAGE and Western blot analysis to confirm full-length protein and assess proteolytic fragments

• Purity assessment: Analytical chromatography methods to ensure consistent purity across batches

• Activity testing: Bioassays using NIH/3T3 mouse embryonic fibroblasts with expected ED50 of 0.05-0.5μg/ml in the presence of appropriate co-factors

• Endotoxin levels: LAL testing to ensure preparations meet endotoxin specifications for research applications

• Glycosylation analysis: Mass spectrometry or lectin-based assays to characterize post-translational modifications

• Stability monitoring: Real-time and accelerated stability studies to establish shelf-life and optimal storage conditions

• Batch consistency: Lot-to-lot comparison of all parameters to ensure reproducible research results