GDF15 Human, His

What is GDF15 and why is it studied with a histidine tag?

GDF15 functions as a mitokine (mitochondrial stress-induced cytokine) that is upregulated under a broad spectrum of conditions including endurance exercise, mitochondrial dysfunction, cellular injury, and inflammation . It plays a critical role in energy homeostasis through activation of the glial-derived neurotrophic factors receptor-α-like (GFRAL) in the hindbrain .

The addition of a histidine tag to recombinant GDF15 serves multiple methodological purposes:

• Enables efficient purification through metal affinity chromatography

• Facilitates detection in experimental systems using anti-His antibodies

• Allows for controlled experiments investigating GDF15's biological activities

The His-tagged version typically contains the mature peptide region (amino acids 197-308 in human GDF15), which represents the active circulating form of the protein .

How is recombinant human GDF15 with His tag produced and characterized?

Production of His-tagged human GDF15 primarily utilizes bacterial expression systems, with specific characteristics:

• Expression system: Typically produced in Escherichia coli (E. coli)

• Protein segment: Contains amino acids Ala197-Ile308 of mature human GDF15

• Tag position: His tag is commonly placed at the N-terminus

• Molecular weight: Calculated MW of 14.2 kDa

• Migration pattern: Migrates as 15 kDa under reducing conditions and 27 kDa under non-reducing conditions (indicating homodimer formation)

• Purity assessment: >90-95% as determined by SDS-PAGE and/or HPLC

• Sterility: Typically 0.22 μm filtered

• Endotoxin levels: Less than 1 EU per μg by the LAL method for research-grade protein

The protein may be provided in solution or lyophilized form with protectants such as trehalose .

How should GDF15-His protein be stored and handled for optimal stability?

Proper storage and handling of GDF15-His protein is critical for maintaining its biological activity:

• Long-term storage: Store in lyophilized state at -20°C or lower

• Reconstitution: Follow specific protocols provided in the Certificate of Analysis

• Post-reconstitution: Aliquot and store at -80°C to minimize freeze-thaw cycles

• Freeze-thaw sensitivity: Avoid repeated freeze-thaw cycles as they can lead to protein degradation

• Working solutions: For short experiments, can be kept at 4°C for limited periods

When planning experiments, researchers should reconstitute only the amount needed for immediate use and store remaining lyophilized protein under recommended conditions.

What are the typical methods to verify GDF15-His protein functionality?

Verification of biological activity for GDF15-His protein can be performed through several approaches:

• Receptor binding assays: Immobilized Human GDF15 with His tag can bind Human GFRAL with an EC50 of approximately 22.8 ng/mL in ELISA-based assays

• Affinity measurements: Surface Plasmon Resonance (SPR) assays can determine binding kinetics, with affinity constants around 0.014 nM reported

• Cell-based assays: Functional studies using cells expressing the GFRAL-RET receptor complex

• Structural analysis: Verification of proper folding through circular dichroism or thermal shift assays

• Bioactivity verification: Testing for expected biological effects like reduced food intake in appropriate model systems

How do measurement approaches for GDF15 differ in research settings?

Recent research highlights important considerations in GDF15 measurement approaches:

• Total vs. H-specific GDF15 measurement:

  • Total GDF15: Detects all GDF15 variants irrespective of genetic variation

  • H-specific GDF15: Detects GDF15 only in the absence of the H202D-variant

This distinction is critical because:

• Both total and H-specific GDF15 increase with acute starvation

• Total GDF15 increases with chronic energy deprivation regardless of leptin repletion

• The H202D variant appears to alter GDF15 associations with metabolites and lipids during metabolic stress

Researchers should consider genetic variants when selecting detection methods and interpreting GDF15 data to avoid methodological artifacts.

How do different energy states affect GDF15 expression and what implications does this have for experimental design?

GDF15 levels change dynamically with various energy states, which researchers must consider when designing experiments:

These findings suggest that:

• Nutritional status must be carefully controlled and documented in GDF15 research

• Baseline measurements and appropriate control groups are essential

• Timing of sample collection is critical for capturing relevant GDF15 dynamics

What methodological approaches should be used to study GDF15 in the context of energy homeostasis?

When investigating GDF15's role in energy homeostasis, researchers should employ comprehensive methodological approaches:

• Dosage selection:

  • Physiological range: 0.2-1.2 ng/mL (healthy humans)

  • Elevated states: 2-4 ng/mL (metabolic stress conditions)

  • Pharmacological: 5-50 ng/mL (for receptor activation studies)

• Administration routes:

  • Central: Direct delivery to brain regions expressing GFRAL

  • Peripheral: Intravenous, intraperitoneal, or subcutaneous depending on research question

• Experimental timeline:

  • Acute effects: 0-24 hours

  • Subchronic effects: 1-7 days

  • Chronic effects: >7 days

• Relevant measurements:

  • Appetite and food intake assessments

  • Body weight and body composition tracking

  • Energy expenditure measurements

  • Tissue-specific GDF15 expression analysis

  • GFRAL-RET signaling pathway activation

  • Relevant metabolic parameters (glucose, insulin, leptin)

How does GDF15 function in the mitochondrial stress response and what methods best capture this relationship?

GDF15 serves as a mitokine reflecting or mediating metabolic stress responses:

• Key findings:

  • GDF15 increases under conditions of energy deprivation in humans

  • Baseline GDF15 positively correlates with triglyceride-rich particles and lipoproteins

  • GDF15 may function as a mitokine signaling mitochondrial stress rather than primarily as a weight loss factor

• Recommended methodological approaches:

  • Mitochondrial function assessment:

    • Oxygen consumption rate measurements

    • Mitochondrial membrane potential analysis

    • ATP production quantification

    • Mitochondrial reactive oxygen species detection

  • Stress pathway monitoring:

    • Integrated stress response (ISR) activation markers

    • Unfolded protein response (UPR) component analysis

    • Analysis of other mitochondrial stress proteins in parallel with GDF15

  • Genetic manipulation experiments:

    • GDF15 knockout and overexpression models

    • Mitochondrial stress induction with and without GDF15 signaling

How can researchers distinguish between endogenous GDF15 and exogenous GDF15-His in experimental systems?

Differentiating endogenous from exogenously added GDF15-His is methodologically important:

• Western blotting: The His-tagged protein shows a slight molecular weight shift compared to endogenous GDF15

• Immunoprecipitation: Use anti-His antibodies to selectively isolate the tagged protein

• ELISA development: Create sandwich ELISAs using anti-His capture and anti-GDF15 detection antibodies

• Mass spectrometry: Can definitively identify tagged versus untagged proteins based on peptide mass differences

• Expression analysis: Monitor GDF15 mRNA to track endogenous production versus protein supplementation

• Temporal analysis: Establish baseline endogenous levels before adding exogenous protein

What is the evolutionary significance of GDF15 and how might this influence experimental approaches?

Understanding GDF15's evolutionary context provides important insights for research design:

• GDF15 likely evolved in the common ancestor of jawed vertebrates, with no clear orthologues in hagfish, lampreys, or lower vertebrates

• Orthologues exist in mammals, reptiles, amphibians, bony fish, and birds with high conservation in the C-terminal region (mature peptide)

• Propeptide conservation is considerably lower, indicating significant remodeling during evolution

• In placental mammals, GDF15 lacks the conserved N-terminal "straitjacket" helix present in canonical TGF-β family members

• This evolutionary simplification suggests that human GDF15 circulates as an active homodimer rather than in a latent complex

Methodological implications:

• Cross-species comparisons should focus on the mature peptide region

• Different experimental approaches may be needed for studies in non-mammalian versus mammalian systems

• When developing neutralizing antibodies or antagonists, target the mature peptide region

How does the GDF15-GFRAL-RET signaling system function and what are key considerations for receptor binding studies?

The discovery that GDF15's receptor is a GFRAL-RET heterodimer with highly specific expression has transformed the understanding of this signaling pathway:

• GFRAL is expressed solely in the hindbrain

• Activation of this receptor results in reduced food intake and weight loss

• This activation is perceived and recalled by animals as aversive

Methodological approaches for receptor studies:

• Binding assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics

  • ELISA-based approaches using immobilized GDF15 and soluble GFRAL

  • Cell-based binding assays with fluorescently-labeled GDF15

• Functional assays:

  • RET phosphorylation detection

  • Downstream signaling pathway activation (MAPK, Akt)

  • Gene expression changes in GFRAL-expressing neurons

  • Behavioral assessments following central administration

• Controls and validation:

  • Use GDF15 antagonists or blocking antibodies

  • Include GFRAL/RET knockout models

  • Compare wild-type GDF15 with mutant versions

  • Consider His-tag position effects on receptor interaction

What are the emerging therapeutic implications of GDF15 research and related methodological considerations?

Recent findings have highlighted potential therapeutic applications of GDF15 pathway modulation:

• GDF15-GFRAL antagonism is emerging as a therapeutic strategy for anorexia/cachexia syndromes

• Metformin elevates circulating GDF15 chronically in humans, and the weight loss caused by this drug appears to be dependent on GDF15

• The human trophoblast produces large amounts of GDF15 from early pregnancy, potentially to encourage avoidance of teratogens

Methodological considerations for therapeutic research:

• Target validation:

  • Genetic loss-of-function and gain-of-function studies

  • Pharmacological inhibition and activation approaches

  • Comparative efficacy studies against established agents

• Biomarker development:

  • GDF15 as a biomarker for therapeutic response

  • Development of robust, standardized GDF15 assays

  • Identification of patient populations most likely to benefit

• Safety assessment:

  • Evaluation of on-target effects in non-CNS tissues

  • Monitoring for potential developmental effects

  • Assessment of impact on stress responses and mitochondrial function