Recombinant Human Fibroblast growth factor 1 protein (FGF1) (Active)

What is Recombinant Human FGF1 protein and what are its alternative designations?

Recombinant Human Fibroblast Growth Factor 1 (FGF1) is a full-length human protein typically expressed in Escherichia coli with >98% purity suitable for various experimental applications including SDS-PAGE, Western Blotting, and functional studies . FGF1 is also known by several alternative designations in the scientific literature, including:

• FGFA

• Acidic fibroblast growth factor (aFGF)

• Endothelial cell growth factor (ECGF)

• Heparin-binding growth factor 1 (HBGF-1)

Understanding these alternative designations is crucial when conducting comprehensive literature searches during experimental design phases.

What are the primary biological functions of FGF1?

FGF1 plays multiple critical roles in cellular processes, functioning as:

• A regulator of cell survival mechanisms

• A mediator of cell division

• A promoter of angiogenesis (formation of new blood vessels)

• A controller of cell differentiation pathways

• A director of cell migration

In vitro, FGF1 functions as a potent mitogen, stimulating cell proliferation. At the molecular level, FGF1 acts as a ligand for fibroblast growth factor receptor 1 (FGFR1) and various integrins . Recent research has also uncovered its potential role in metabolism regulation, particularly in glucose homeostasis .

How does FGF1's signaling mechanism function at the molecular level?

FGF1 signaling involves a complex molecular cascade:

• Receptor binding: FGF1 binds to FGFR1 in the presence of heparin, which facilitates FGFR1 dimerization .

• Receptor activation: This dimerization triggers sequential autophosphorylation on tyrosine residues of FGFR1 .

• Signaling cascade initiation: The phosphorylated residues serve as docking sites for interacting proteins .

• Downstream activation: This process induces the phosphorylation and activation of multiple downstream targets including FGFR1, FRS2, MAPK3/ERK1, MAPK1/ERK2, and AKT1 .

• Integrin-mediated signaling: FGF1 also binds to integrin ITGAV:ITGB3, forming a ternary complex with integrin and FGFR1, recruiting PTPN11 to the complex—a process essential for FGF1 signaling .

This dual-receptor interaction mechanism explains the diverse biological effects of FGF1 in different cellular contexts.

What is the optimal expression system for producing active recombinant FGF1?

E. coli remains the preferred expression system for recombinant FGF1 production. The recommended protocol based on current research includes:

• Bacterial strain selection: Use E. coli BL21 (DE3) pLysS expression strain .

• Growth conditions optimization:

  • Grow bacteria in LB medium with 100 μg/mL ampicillin at 37°C

  • Maintain shaking at 250 rpm until OD600 reaches 0.9

  • Reduce temperature to 25°C for protein induction

  • Add IPTG to a final concentration of 0.5 mM

  • Continue expression for 12 hours

This approach consistently yields high levels of soluble, properly folded FGF1 protein suitable for research applications.

What is the recommended purification protocol for obtaining high-purity FGF1?

A multi-step purification protocol utilizing FGF1's high affinity for heparin yields optimal results:

• Cell lysis preparation:

  • Harvest cells by centrifugation at 3000g for 20 minutes

  • Wash once with PBS

  • Resuspend in buffer A (20 mM Tris, 0.5 M NaCl, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF, pH 7.4)

  • Sonicate for 5 minutes in 4 rounds

• Initial clarification:

  • Remove cell debris via ultracentrifugation at 20,000g at 4°C for 45 minutes

• Affinity chromatography:

  • Load clarified lysate onto a heparin-sepharose gravity column

  • Incubate at 4°C for 1 hour

  • Wash with buffer B (20 mM Tris, 0.7 M NaCl, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF, pH 7.4) for 4 column volumes

  • Elute protein with buffer C (20 mM Tris, 2 M NaCl, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF, pH 7.4)

This protocol consistently yields recombinant FGF1 with purity exceeding 98%, making it suitable for sensitive research applications.

What analytical methods should be used to verify recombinant FGF1 identity and activity?

Multiple complementary analytical techniques should be employed to thoroughly characterize recombinant FGF1:

• Purity assessment:

  • SDS-PAGE for molecular weight confirmation and purity estimation

  • Western blotting using anti-FGF1 antibodies for identity verification

• Structural integrity confirmation:

  • Liquid chromatography-mass spectrometry (LC-MS) to verify molecular weight and detect potential modifications

  • Correct molecular weight for native FGF1 is approximately 15,300.8 Da

  • Modified constructs (e.g., FGF1-DOTA-Tb) should show expected mass increases (683.1 Da for one maleimide-DOTA molecule and one Tb cation)

• Functional analysis:

  • Cell proliferation assays using FGF1-responsive cell lines

  • Receptor binding assays to confirm interaction with FGFR1

  • Phosphorylation analysis of downstream targets (MAPK3/ERK1, MAPK1/ERK2, AKT1)

This multi-parameter approach ensures both structural and functional integrity of the recombinant protein.

How does FGF1's thermodynamic stability impact its biological activity?

FGF1's intrinsic thermodynamic stability directly influences its biological function:

• Baseline stability: Native FGF1 has relatively low thermodynamic stability with a denaturation temperature close to physiological conditions .

• Stability-activity relationship: Research has demonstrated that increased thermodynamic stability of FGF1 can effectively compensate for decreased heparin binding in:

  • FGFR activation

  • Induction of DNA synthesis

  • Cell proliferation

• Research application: This principle has been experimentally validated through the progressive introduction of stabilizing mutations into the K118E (K132E) FGF1 variant, which initially showed reduced affinity for heparin and was inactive in stimulating DNA synthesis. Through strategic stabilization, full mitogenic activity was restored despite impaired heparin binding .

This finding has significant implications for designing FGF1 variants with modified stability profiles for specific research applications.

What is the relationship between heparin binding and FGF1 function?

Heparin plays a critical but potentially compensable role in FGF1 function:

• Stabilization effect: Binding to heparin significantly increases FGF1's stability, traditionally considered essential for the formation of active FGF1-FGFR complexes .

• Complex formation: Heparin facilitates the dimerization of FGFR1 upon FGF1 binding, leading to receptor activation .

• Stability-binding tradeoff: Experimental evidence indicates that increased thermodynamic stability of FGF1 can compensate for decreased heparin binding in receptor activation and subsequent biological functions .

• Methodological implications: When designing experiments with FGF1, researchers should consider including heparin (typically 10 U/mL heparin sulfate) in cell culture media to optimize FGF1 activity, particularly when using non-stabilized variants .

This stability-binding relationship provides a conceptual framework for understanding FGF1 variants with altered heparin-binding properties.

How can engineered FGF1 variants be designed for research applications?

Strategic engineering of FGF1 can create variants with specialized properties for different research applications:

• Site-specific modifications:

  • C-terminal additions of functional groups (e.g., free thiol) enable precise conjugation of molecules like maleimide-DOTA

  • Michael-type addition reactions can be used for conjugating various molecules to engineered FGF1

• Stability engineering:

  • Variants with increased thermodynamic stability can be created to compensate for decreased heparin binding

  • Progressive introduction of stabilizing mutations can restore activity in otherwise inactive variants

• Conjugation validation:

  • Confirm successful conjugation through multiple methods:

    • Gel electrophoresis for mobility shifts

    • LC-MS for precise molecular weight determination

    • For radioactive conjugates, thin-layer chromatography with phosphor imaging (showing >90% conjugation efficiency for properly designed constructs)

These engineering approaches expand FGF1's utility in diverse research applications from imaging to targeted therapy.

How is FGF1 being applied in diabetes research?

FGF1 has emerged as a promising research target in diabetes studies, with several key findings:

• Central nervous system effects:

  • Intracerebroventricular injection of FGF1 in rat models of type 2 diabetes mellitus (T2DM) induces sustained remission of hyperglycemia

  • A single dose delivered directly to the brain causes sustained diabetes remission across multiple rodent models

• Insulin-independent mechanism:

  • FGF1's glucose-lowering effects appear to be independent of insulin-dependent glucose clearance

  • In Zucker diabetic fatty fa/fa (ZDF) rats, a model closely approximating human T2DM:

    • FGF1 treatment delayed the decline in β-cell function

    • β-cell mass increased 3 weeks after injection compared to control animals

    • After 7 weeks, β-cell mass decreased to match control values

  • Intravenous insulin injection had reduced glucose-lowering effects in FGF1-treated rats compared with controls

• Potential mechanism:

  • Evidence suggests hepatic glucose uptake, which is insulin-independent, may be a possible mechanism contributing to FGF1's antidiabetic effects

These findings highlight FGF1 as a valuable research tool for exploring novel mechanisms in glucose regulation and potential therapeutic approaches for diabetes.

What methods are effective for studying FGF1 translocation across cellular membranes?

To investigate FGF1's cellular trafficking and translocation, researchers can employ these methodological approaches:

• Cell fractionation technique:

  • Plate cells at 5×10^5 cells per well and culture for 2 days

  • Shift to serum-free medium for 24 hours

  • For inhibition studies, pretreat cells with inhibitors (e.g., 100 μM bafilomycin, 10 nM radicicol, or 10 μM SB203590) 20 minutes before FGF1 treatment

  • Incubate with labeled FGF1 (e.g., 300 ng/mL eFGF1-161Tb) in complete media containing 10 U/mL heparin for 6 hours

  • Wash cells with PBS buffer 3 times

  • Lyse with 500 μL nucleus collection buffer (0.1 M NaCl, 10 mM Na2HPO4, 1% Triton X-100, 1 mM EDTA)

  • Separate nuclei from cytoplasmic fraction by centrifugation at 20,000g for 2 minutes

  • Wash nuclei once with lysis buffer

  • Measure radioactivity of each fraction with a gamma counter

• Visualization of cellular uptake:

  • Plate cells on coverslips at 5×10^5 cells per well

  • After 24 hours, treat with fluorescently labeled FGF1 (e.g., FGF1-DOX) at concentrations of 1-10 μM in complete media containing 10 U/mL heparin

  • Incubate at 37°C for 2 hours

  • Wash with PBS and fix in 4% formaldehyde for 15 minutes

  • Counterstain nuclei with Hoechst 33342

  • Mount coverslips with antifade mounting medium

  • Image using confocal microscopy (e.g., with 63× objective)

These complementary approaches provide quantitative and qualitative data on FGF1's translocation behavior.

How can FGF1 be engineered for targeted therapy applications?

Engineering FGF1 for targeted therapeutic applications involves several strategic considerations:

• Conjugation strategies:

  • Site-specific modification at the C-terminal end of engineered FGF1 (eFGF1) provides a controlled approach

  • Michael-type addition reactions between maleimide-containing molecules and free thiol on eFGF1 enable precise conjugation

• Radioisotope conjugation:

  • For targeted radiotherapy applications, FGF1 can be conjugated with radioisotopes such as 161Tb

  • The process involves:

    • Linking maleimide-DOTA to eFGF1

    • Incubating eFGF1-DOTA with Tb3+ in pH 6.0 buffer at 37°C for 16 hours (mild pH conditions prevent FGF1 unfolding)

    • Achieving conjugation efficiency exceeding 90%

• Verification techniques:

  • Gel electrophoresis to confirm conjugate formation

  • LC-MS to verify exact molecular weight changes (e.g., increase of 683.1 Da for one maleimide-DOTA molecule and one Tb cation)

  • For radioactive conjugates, thin-layer chromatography with phosphor imaging

• Target validation:

  • Structural modeling of the dimeric 2:2:2 FGF1:FGFR1:Heparin ternary complex helps predict binding interactions

  • Cell uptake studies with engineered conjugates confirm targeting specificity

These approaches enable the development of FGF1-based targeted therapeutic agents for research applications.

What genetic variations in the FGF1 pathway are associated with disease states?

Genetic studies have revealed significant associations between FGF1 pathway genes and disease states, particularly hypertension:

• FGF1 genetic associations:

  • Variants in the FGF1 gene co-segregate with familial susceptibility to hypertension

  • Glomerular upregulation of FGF1 is associated with hypertension

• FGFBP1 associations:

  • Family-based analysis showed that parents transmitted the major allele of the rs16892645 polymorphism in the FGFBP1 gene to hypertensive offspring more frequently than expected by chance (P = 0.005)

  • This association was validated in an independent cohort of 807 unrelated Polish subjects

  • Compared with normotensive subjects, hypertensive subjects had approximately:

    • 1.5-fold higher expression of renal FGFBP1 mRNA (P = 0.04)

    • 1.4-fold higher expression of renal FGFBP1 protein (P = 0.001)

• Localization of expression changes:

  • By immunohistochemistry, hypertension-related upregulation of FGFBP1 was most apparent in the glomerulus and juxtaglomerular space

These findings highlight the value of systematic genetic analysis in identifying previously undescribed genetic associations in the FGF1 signaling pathway.

What methodological approaches are recommended for studying FGF1 pathway genetics?

Researchers investigating genetic associations in the FGF1 pathway should consider these methodological approaches:

• SNP selection for comprehensive pathway coverage:

  • Select SNPs to achieve dense coverage for each gene in the pathway

  • Aim for high proportion of common genetic variance explained (>90%)

  • Use SNPs with high maximum r^2 values (>0.9)

• Quality control criteria:

  • Exclude SNPs with genotyping rate <90%

  • Exclude SNPs violating Hardy-Weinberg equilibrium (P < 0.01)

  • Exclude SNPs with minor allele frequency <5%

• Family-based association testing:

  • Use family-based association test (FBAT) for initial analysis in family cohorts

  • Apply false positive discovery rate correction for multiple testing

• Replication in independent cohorts:

  • Validate findings in biologically unrelated subjects

  • Use age-, gender-, and body mass index–adjusted logistic regression models under additive model of inheritance

• Combined analysis approaches:

  • Use generalized estimating equations–based approaches to extract information from both unrelated individuals and different types of nuclear families

  • This method integrates principal component analysis with transmission disequilibrium test strategies to minimize potential stratification effects

  • Improves power to detect genetic associations compared with typical tests used in analysis of family and unrelated samples separately

These methodological considerations provide a robust framework for investigating genetic associations in the FGF1 pathway.

What are the critical factors for successful FGF1 functional assays?

When designing functional assays with FGF1, several critical factors should be considered:

• Heparin supplementation:

  • Include 10 U/mL heparin sulfate in cell culture media when testing FGF1 activity

  • This is essential for optimal FGFR1 dimerization and activation

• Stability considerations:

  • Native FGF1 has relatively low thermodynamic stability with denaturation temperature close to physiological conditions

  • For experiments requiring extended incubation periods, consider using stabilized FGF1 variants

• Concentration optimization:

  • Typical working concentrations range from:

    • 1-10 μM for cellular uptake studies

    • 300 ng/mL for translocation studies

• Control experiments:

  • Include translocation inhibitors (bafilomycin, radicicol, SB203590) as negative controls for mechanistic studies

  • These inhibitors should be added 20 minutes before FGF1 treatment

• Cell type selection:

  • Different cell types express varying levels of FGFR1 and relevant integrins

  • Preliminary experiments should validate receptor expression in selected cell lines

Attention to these factors will improve reliability and reproducibility of FGF1 functional assays.

How can researchers effectively study the interplay between FGF1 and its binding partners?

To investigate interactions between FGF1 and its binding partners, researchers should employ these approaches:

• Structural modeling:

  • Computational modeling of the dimeric 2:2:2 FGF1:FGFR1:Heparin ternary complex provides insights into binding interactions

  • Structural predictions can guide mutagenesis strategies

• Binding assays:

  • FGF1 binds to FGFR1 in the presence of heparin

  • Also binds to integrin ITGAV:ITGB3

  • Forms a ternary complex with integrin and FGFR1, with recruitment of PTPN11

• Signaling cascade analysis:

  • Monitor phosphorylation and activation of downstream targets:

    • FGFR1

    • FRS2

    • MAPK3/ERK1

    • MAPK1/ERK2

    • AKT1

• Compensation mechanisms:

  • Investigate how increased stability can compensate for decreased heparin binding

  • Design step-wise mutation studies to isolate effects of individual binding parameters

These methodological approaches provide comprehensive insights into the complex interplay between FGF1 and its multiple binding partners.

What are the most common technical challenges when working with recombinant FGF1?

Researchers should be aware of these common technical challenges when working with recombinant FGF1:

• Stability limitations:

  • Native FGF1 has relatively low thermodynamic stability with denaturation temperature close to physiological conditions

  • Storage and handling conditions are critical for maintaining activity

• Heparin dependency:

  • FGF1 activity is highly dependent on heparin presence

  • Insufficient heparin can lead to false negative results in functional assays

• pH sensitivity:

  • FGF1 unfolds at low pH, limiting buffer options

  • When conjugating FGF1 to other molecules, maintain pH ≥6.0 to prevent unfolding

• Expression challenges:

  • Temperature control during expression is critical (reduce to 25°C for protein induction)

  • Appropriate strain selection (E. coli BL21 (DE3) pLysS) improves yield

• Purification considerations:

  • High salt concentration (2M NaCl) required for efficient elution from heparin columns

  • Multiple additives (DTT, EDTA, PMSF) needed in buffers for stability

Awareness of these technical challenges and their solutions can significantly improve experimental outcomes when working with recombinant FGF1.