Bet v 2.0101

What is Bet v 2.0101 and why is it significant for allergen research?

Bet v 2.0101 (also known as Profilin-1) is a recombinant allergen protein from Betula verrucosa (silver birch) pollen. It represents a clinically relevant allergen that binds to IgE-type human antibodies and plays a significant role in allergic sensitization . As a member of the profilin family, Bet v 2.0101 serves as an important model for studying cross-reactivity patterns in plant allergies, particularly in patients with birch pollen allergies who may experience oral allergy syndrome with certain foods .

How does Bet v 2.0101 compare to other major birch pollen allergens?

While Bet v 1 is considered the major birch pollen allergen (affecting approximately 5% of the Austrian population according to studies), Bet v 2.0101 represents another important allergen with distinct structural and immunological properties . Unlike Bet v 1, which belongs to the PR-10 protein family, Bet v 2.0101 is a profilin that demonstrates significant structural homology with profilins from other plant species such as Amb a 8.0101, Art v 4.0101, and Phl p 12.0101 . This structural similarity is responsible for the cross-reactivity observed between birch pollen and various plant food allergies.

What are the key structural characteristics of Bet v 2.0101?

Bet v 2.0101 is a relatively small protein with the following biochemical parameters:

• Molecular weight: Reported as 11 kDa to 16.2 kDa (variation likely due to purification tags)

• Isoelectric point: pH 5.67

• Secondary structure composition (based on crystal structure analysis): α-helices 24%, β-strands 35%, and random coils 41%

The protein structure has been extensively characterized through crystallography and spectroscopic methods, revealing structural elements that contribute to its allergenicity and cross-reactivity with other profilins .

How can thermal stability studies inform our understanding of Bet v 2.0101?

Thermal stability studies, particularly those using differential scanning fluorimetry (DSF), have revealed that Bet v 2.0101 exhibits distinct stability profiles compared to other allergenic profilins. When compared with Art v 4.0101, Amb a 8.0101, and other profilins, Bet v 2.0101 shows melting temperatures that are 8-19°C lower, with the greatest differences observed at pH 4.0 . These stability differences may influence the protein's resistance to degradation in various physiological environments, potentially affecting its allergenicity profile and experimental handling requirements.

What expression systems are most effective for recombinant Bet v 2.0101 production?

Escherichia coli represents the predominant expression system for recombinant Bet v 2.0101 production across research laboratories . This bacterial expression system offers advantages including:

• High protein yields (comparable to other profilins like Cuc m 2.0101, which yields up to 40 mg/L culture)

• Consistent protein folding and structure formation

• Compatibility with various purification tags (including Strep tags and poly-histidine tags)

When using E. coli expression systems, researchers typically achieve protein recovery rates of approximately 80% after purification tag cleavage, making this an efficient approach for obtaining research-grade Bet v 2.0101 .

What purification strategies yield the highest purity Bet v 2.0101 for experimental applications?

Multiple purification approaches have proven effective for obtaining high-purity Bet v 2.0101:

• Sequential chromatography combining ion exchange and affinity chromatography has demonstrated success in producing preparations with >95% purity as verified by Coomassie Brilliant Blue stained SDS-PAGE .

• Affinity chromatography utilizing the protein's fusion tags (such as deca-histidine or Strep tags) followed by size exclusion chromatography (SEC) to remove aggregates .

The choice of purification strategy should consider the intended experimental application, with structural studies typically requiring the highest purity levels (>95%), while some immunological assays may tolerate slightly lower purity (>80%) .

How does Bet v 2.0101 interact with cytoskeletal components in cellular systems?

Bet v 2.0101, like other profilins, exhibits concentration-dependent effects on actin cytoskeleton dynamics. At high concentrations, it prevents actin polymerization, while at low concentrations, it augments polymerization . This concentration-dependent effect is critical for researchers using Bet v 2.0101 in cellular studies, as experimental concentration must be carefully controlled to achieve desired cytoskeletal effects.

Additionally, Bet v 2.0101 inhibits the creation of inositol trisphosphate (IP3) and diacylglycerol (DG) by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) . This interaction with cellular signaling pathways provides further research opportunities for understanding the biological effects of this allergen beyond its allergenic properties.

What is the immunological significance of Bet v 2.0101 in allergic responses?

Bet v 2.0101 functions as an allergen by binding to IgE-type human antibodies, particularly in patients sensitized to birch pollen . Research has demonstrated that Bet v 2.0101 can be used in diagnostic applications, including ELISA assays designed to detect allergen-specific human plasma IgG4 antibodies from Betula verrucosa-positive donors .

The protein shows significant cross-reactivity with other plant profilins. Studies using ELISA inhibition assays have demonstrated IgE reactivity to multiple profilins (Amb a 8.0101, Art v 4.0101, Bet v 2.0101, and Phl p 12.0101) in allergic patients, highlighting the pan-allergenic nature of this protein family .

What analytical techniques are most effective for studying Bet v 2.0101 structure-function relationships?

Multiple complementary analytical approaches have been successfully employed to characterize Bet v 2.0101:

• X-ray crystallography: Has been used to determine high-resolution structures, providing insights into the protein's three-dimensional conformation .

• Circular dichroism (CD) spectroscopy: Effective for analyzing secondary structure composition and conformational changes under different conditions .

• Differential scanning fluorimetry (DSF): Valuable for thermal stability assessments and comparative studies with other profilins .

• Flow cytometry: Used for functional studies, particularly to evaluate activation of basophils and lymphocytes from allergic donors in response to Bet v 2.0101 stimulation .

• Western blotting and immunodot analyses: Employed for detecting binding interactions with patient-derived antibodies .

How should researchers optimize storage conditions for maintaining Bet v 2.0101 stability?

Based on experimental data, the following storage recommendations maximize Bet v 2.0101 stability:

• Buffer composition: Neutral to slightly alkaline pH with 20% glycerol as a cryoprotective agent , or alternatively, 100mM Tris Buffer, pH 8, with 150mM NaCl .

• Storage temperature: -70°C or below for long-term storage , with -20°C to -80°C suitable for reconstituted protein .

• Physical state: Maintain as lyophilized product until needed; reconstituted protein should be used immediately or aliquoted to prevent freeze-thaw cycles .

• Sterility: Use 0.2 μm filter sterilization for solutions intended for cell-based applications .

• Avoid freeze/thaw cycles as they significantly compromise protein integrity and biological activity .

How does the thermal stability of Bet v 2.0101 compare with other allergenic profilins?

Comparative thermal stability studies using differential scanning fluorimetry (DSF) have revealed significant differences between allergenic profilins:

The experimental data demonstrate that Bet v 2.0101 exhibits lower thermal stability than Cuc m 2.0101, with the largest stability differences observed at acidic pH (pH 4.0) . These stability differences correlate with computational predictions and may influence the protein's behavior in experimental systems, particularly those that involve pH changes or thermal stress.

What sequence homologies exist between Bet v 2.0101 and other allergenic profilins?

Bet v 2.0101 shares significant sequence homology with profilins from various plant species. Sequence alignment studies have demonstrated the following relationships:

• High sequence conservation exists between Bet v 2.0101 and other allergenic profilins including Amb a 8.0101, Art v 4.0101, and Phl p 12.0101 .

• This sequence conservation translates to structural similarity, explaining the observed cross-reactivity patterns in allergic patients.

• Despite high sequence homology, specific amino acid differences in key regions likely account for the measured differences in thermal stability and potentially influence epitope recognition by patient antibodies .

The sequence conservation among profilins explains why patients sensitized to birch pollen often demonstrate allergic reactions to multiple plant species, making Bet v 2.0101 an important model allergen for studying cross-reactivity mechanisms .

How can molecular dynamics simulations enhance our understanding of Bet v 2.0101 allergenicity?

Molecular dynamics (MD) simulations represent a powerful computational approach for investigating Bet v 2.0101 allergenicity beyond static structural data. MD simulations can provide insights into:

• Conformational flexibility and the identification of dynamic epitopes that may not be apparent in static crystal structures.

• Protein-protein interaction interfaces between Bet v 2.0101 and IgE antibodies, potentially revealing key binding determinants.

• Effects of environmental conditions (pH, temperature, ionic strength) on protein structure and stability, complementing experimental thermal stability data .

• Ligand binding dynamics, particularly interactions with actin and PIP2, which could influence the protein's allergenic properties in cellular contexts .

These computational approaches, when integrated with experimental data, can guide epitope mapping studies and inform the design of hypoallergenic variants for immunotherapy applications.

What methodological approaches can resolve contradictions in reported biochemical parameters of Bet v 2.0101?

The scientific literature contains some discrepancies in the reported biochemical parameters for Bet v 2.0101, particularly regarding molecular weight (reported as both 11 kDa and 16.2 kDa ). To resolve these contradictions, researchers should:

• Carefully account for the contribution of purification tags to molecular weight calculations (Strep tags, His tags, etc.).

• Employ multiple analytical techniques for molecular weight determination:

  • Mass spectrometry (particularly ESI-MS) for precise mass determination

  • SDS-PAGE calibrated with appropriate molecular weight standards

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

• Characterize the exact amino acid sequence of the recombinant construct, including any non-native residues remaining after tag cleavage.

• Report protein parameters with explicit descriptions of the construct used, including the presence or absence of tags and any modifications to the native sequence.

These methodological considerations ensure experimental reproducibility and facilitate meaningful comparisons across different research studies.

What emerging technologies offer new insights into Bet v 2.0101 structure-function relationships?

Several cutting-edge technologies show promise for advancing our understanding of Bet v 2.0101:

• Cryo-electron microscopy: May reveal structural details of Bet v 2.0101 in complex with antibodies or cellular receptors at near-atomic resolution.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Can provide insights into protein dynamics and conformational changes upon ligand binding.

• Single-molecule FRET techniques: Offer the potential to observe real-time conformational changes during interactions with binding partners.

• Advanced computational approaches: Including machine learning algorithms for predicting epitopes and allergenicity profiles based on sequence and structural data.

These methodologies, when applied to Bet v 2.0101 research, will likely expand our understanding of this allergen's role in allergic disease and potentially inform novel therapeutic strategies.

How might recombinant Bet v 2.0101 contribute to personalized allergy diagnostics and therapeutics?

Recombinant Bet v 2.0101 has significant potential for advancing personalized approaches to allergy management:

• Component-resolved diagnostics: Purified Bet v 2.0101 enables precise determination of sensitization profiles in allergic patients, distinguishing between sensitization to different birch pollen components.

• Biomarker development: Quantitative measurement of Bet v 2.0101-specific antibodies may serve as biomarkers for monitoring treatment responses.

• Epitope-focused immunotherapy: Identification of immunodominant epitopes could guide the development of peptide-based therapies targeting specific regions of Bet v 2.0101.

• Hypoallergenic variants: Structure-based design of modified Bet v 2.0101 molecules with reduced IgE binding but preserved T-cell epitopes could enhance the safety profile of immunotherapy approaches.