Aln G 4.0101

What is Aln G 4.0101 and what are its key molecular features?

Aln G 4.0101 is an allergenic protein found in the pollen of Alnus glutinosa (alder) that belongs to the polcalcin family. It comprises two EF-hand motifs that complex calcium ions and induce conformational changes upon binding . This protein is alternatively known as polcalcin Aln g 4 or calcium-binding pollen allergen Aln g 4 .

The recombinant form of Aln G 4.0101 is a glycosylated polypeptide chain with a calculated molecular mass of approximately 10,185 Daltons when produced in SF9 insect cells with a 6xHis tag at the N-terminus . This molecular structure contributes to its allergenic properties and its ability to trigger immune responses in sensitized individuals.

How does the structure of Aln G 4.0101 relate to its biological function?

The defining structural feature of Aln G 4.0101 is its two EF-hand motifs, which are helix-loop-helix structural domains that specifically bind calcium ions . When calcium binds to these EF-hand motifs, it induces conformational changes in the protein structure that are critical to its biological activity.

The calcium-binding capability of Aln G 4.0101 is significant because it can affect:

• Protein stability and folding

• Allergenic epitope presentation

• Cross-reactivity with related allergens

• Interaction with immune system components

These structural properties make Aln G 4.0101 an important model for studying calcium-dependent allergen functions and developing targeted therapeutic approaches for pollen allergies.

What are the recommended methods for recombinant production of Aln G 4.0101?

Recombinant production of Aln G 4.0101 can be achieved using SF9 insect cell expression systems, which allow for proper post-translational modifications including glycosylation . The expression typically includes a 6xHis tag at the N-terminus to facilitate purification .

The recommended expression protocol based on similar allergen production methods involves:

• Transformation of the expression vector containing the Aln G 4.0101 gene into the host system

• Culture growth under optimized conditions (similar to protocols used for Bet v 1-specific nanobodies )

• Induction of protein expression using appropriate inducers

• Cell harvesting by centrifugation (e.g., 15 minutes at 4°C and 2300 g)

• Cell lysis using appropriate buffers (such as TES buffer with additives)

• Protein extraction from the cellular fractions

• Purification via immobilized metal affinity chromatography using nickel-based resins

• Dialysis against appropriate buffers (such as PBS)

• Quality verification using SDS-PAGE and other analytical methods

For optimal yield and purity, additional chromatographic steps may be required based on specific research needs and quality requirements.

What purification techniques are most effective for Aln G 4.0101?

Immobilized metal affinity chromatography (IMAC) represents the primary purification method for His-tagged Aln G 4.0101 . Based on protocols used for similar allergen proteins, the following detailed purification workflow is recommended:

• Prepare nickel affinity gel by washing with equilibration buffer (typically 50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole)

• Incubate the protein extract with the prepared gel (1:1 mixture) for approximately 2 hours at room temperature

• Transfer the mixture to a column for gravity-flow chromatography

• Wash extensively with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole)

• Elute the protein using elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 250 mM imidazole)

• Collect fractions and analyze for protein content

• Dialyze against the final formulation buffer (e.g., 20 mM HEPES buffer pH-7.9)

• Verify purity by SDS-PAGE under both reducing and non-reducing conditions (target purity >80%)

For applications requiring higher purity, additional purification steps such as size exclusion chromatography or ion exchange chromatography may be implemented.

What are the optimal conditions for storing and maintaining Aln G 4.0101?

For optimal stability and activity maintenance, Aln G 4.0101 should be stored according to the following guidelines:

• For short-term storage (2-4 weeks), maintain the protein at 4°C in its formulation buffer

• For long-term storage, keep the protein frozen at -20°C

• Avoid multiple freeze-thaw cycles to prevent protein degradation and activity loss

• Store in the recommended formulation buffer (20 mM HEPES buffer pH-7.9 with 6 M urea)

The presence of 6 M urea in the formulation buffer suggests that the protein may require denaturing conditions for stability, which is an important consideration for experimental design. Researchers should note that the protein's conformational state in this buffer may differ from its native state in biological systems.

How does calcium concentration affect the stability and conformation of Aln G 4.0101?

As a calcium-binding protein with EF-hand motifs, Aln G 4.0101's stability and conformation are directly influenced by calcium concentration. When designing experiments with this allergen, researchers should consider:

• Calcium-free states may present different conformational epitopes than calcium-bound states

• Experimental buffers should have defined and controlled calcium concentrations

• Calcium-binding can influence the protein's resistance to proteolytic degradation

• Structural studies (e.g., circular dichroism or thermal stability assays) should be performed under both calcium-saturated and calcium-free conditions

Researchers investigating the immunological properties of Aln G 4.0101 should carefully account for calcium's effect on the protein's allergenic epitopes and cross-reactivity potential with related allergens.

How does Aln G 4.0101 cross-react with other pollen allergens?

Aln G 4.0101 belongs to the polcalcin family of calcium-binding allergens, which demonstrates significant cross-reactivity across different plant species. Based on research on related allergens, the following cross-reactivity patterns are important to consider:

• Strong cross-reactivity with other tree pollen polcalcins, particularly from botanically related species

• Potential cross-reactivity with polcalcins from:

  • Birch (Bet v 4)

  • Hazel (Cor a 4)

  • Other members of the Betulaceae family

When designing immunological experiments, researchers should include appropriate controls to account for this cross-reactivity. This may involve:

• Using structurally related polcalcins as control antigens

• Including calcium-binding proteins from unrelated sources as negative controls

• Performing competitive inhibition assays to quantify the degree of cross-reactivity

What methodologies are recommended for studying the allergenicity of Aln G 4.0101?

Several methodological approaches can be employed to study the allergenic properties of Aln G 4.0101:

• IgE-binding assays:

  • ELISA-based methods with immobilized Aln G 4.0101 (2 μg/ml in bicarbonate buffer)

  • Immunoblotting using patient sera

  • Basophil activation tests

• Cross-reactivity assessment:

  • Inhibition ELISA with related polcalcins

  • Multi-allergen screening arrays

• Epitope mapping:

  • Synthetic peptide analysis

  • Mutagenesis studies targeting the calcium-binding regions

  • Competitive binding with monoclonal antibodies

• Functional assays:

  • RBL (rat basophilic leukemia) cell degranulation assays

  • Nanobody-based allergen suppression strategies, similar to those used for Bet v 1

• Structural analysis:

  • Circular dichroism to assess secondary structure

  • Differential scanning calorimetry to evaluate thermal stability

When interpreting results, it is important to consider both calcium-bound and calcium-free states of the protein, as these may present different allergenic epitopes.

How can Aln G 4.0101 be used in developing novel allergy diagnostics?

Aln G 4.0101 offers several opportunities for developing next-generation allergy diagnostics:

• Component-resolved diagnostics (CRD):

  • Inclusion of Aln G 4.0101 in multi-allergen arrays

  • Differential diagnosis between primary sensitization and cross-reactivity

• Recombinant allergen-based testing:

  • Use of highly purified Aln G 4.0101 for standardized testing

  • Comparison with extract-based testing for improved specificity

• Conformational epitope analysis:

  • Testing patient IgE reactivity against native versus denatured Aln G 4.0101

  • Calcium-dependent epitope mapping

• Cross-reactivity profiling:

  • Development of inhibition-based assays to determine the primary sensitizing allergen

  • Creation of cross-reactivity maps among calcium-binding pollen allergens

Researchers can implement these approaches using methodologies similar to those employed in studies of Bet v 1-specific nanobodies, which have demonstrated the capacity to suppress polyclonal IgE binding to corresponding allergens .

What are the methodological considerations for studying calcium-dependent conformational changes in Aln G 4.0101?

Investigating the calcium-dependent conformational dynamics of Aln G 4.0101 requires specialized biophysical techniques:

• Structural analysis methods:

  • Circular dichroism spectroscopy to monitor secondary structure changes upon calcium binding

  • Fluorescence spectroscopy to detect tertiary structure alterations

  • Nuclear magnetic resonance (NMR) for atomic-level structural information

• Binding studies:

  • Isothermal titration calorimetry (ITC) to determine calcium binding parameters

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• Computational approaches:

  • Molecular dynamics simulations of calcium binding and resulting conformational changes

  • Homology modeling based on related polcalcins with known structures

• Functional correlation:

  • Analysis of calcium-dependent IgE binding

  • Correlation between calcium occupancy and allergenicity

When designing these experiments, researchers should carefully control buffer conditions, especially calcium concentration, and consider using calcium chelators (EGTA) as negative controls. Experiments should be performed with multiple protein batches to ensure reproducibility, given the potential for batch-to-batch variation in recombinant protein production.

How does Aln G 4.0101 compare structurally and functionally with other pollen allergens?

Aln G 4.0101 can be compared with other pollen allergens based on several parameters:

Key functional differences include:

• Aln G 4.0101 binds calcium ions through its EF-hand motifs, unlike PR-10 proteins

• The calcium-binding property influences its conformational stability and allergenicity

• As a polcalcin, Aln G 4.0101 contributes to cross-reactivity between different pollen sources

• Despite being a minor allergen, its high cross-reactivity makes it clinically relevant

These comparative aspects are important when designing diagnostic panels and interpreting patient sensitization patterns.

What experimental design considerations are needed when comparing Aln G 4.0101 with other Betulaceae allergens?

When conducting comparative studies between Aln G 4.0101 and other Betulaceae allergens, researchers should consider:

• Standardization of experimental conditions:

  • Use the same expression systems for recombinant allergen production

  • Apply consistent purification protocols

  • Ensure comparable protein quality and purity (>80% as determined by SDS-PAGE)

• Appropriate controls:

  • Include non-Betulaceae polcalcins to assess family-specific versus protein family-specific effects

  • Use non-allergenic calcium-binding proteins as functional controls

  • Include Der p 2 (house dust mite) or other unrelated allergens as negative controls

• Analytical considerations:

  • Perform experiments under both reducing and non-reducing conditions

  • Include calcium-supplemented and calcium-depleted conditions

  • Assess native and denatured protein conformations

• Cross-reactivity analysis:

  • Conduct inhibition studies with patient sera

  • Use monoclonal antibodies to identify shared epitopes

  • Perform epitope mapping to identify conserved allergenic determinants

• Data interpretation:

  • Account for differences in protein concentrations

  • Consider the impact of tags and fusion partners on allergenicity

  • Normalize results appropriately for valid comparisons

Following these methodological considerations ensures scientifically sound comparisons between Aln G 4.0101 and other allergens in the Betulaceae family.

What are the emerging research areas for Aln G 4.0101 in allergy treatment?

Several promising research directions for Aln G 4.0101 in allergy treatment include:

• Targeted immunotherapy approaches:

  • Development of hypoallergenic variants through site-directed mutagenesis of calcium-binding sites

  • Creation of chimeric proteins with reduced allergenicity but maintained immunogenicity

• Nanobody-based therapeutic strategies:

  • Development of Aln G 4.0101-specific nanobody trimers, similar to those developed for Bet v 1

  • Evaluation of their capacity to suppress polyclonal IgE binding to the allergen

  • Creation of multivalent constructs targeting multiple epitopes simultaneously

• Epitope-focused vaccine development:

  • Identification of non-IgE binding T cell epitopes for safe immunotherapy

  • Design of peptide-based vaccines targeting these epitopes

• Cross-reactive immunotherapy:

  • Exploration of cross-protective effects when treating with related polcalcins

  • Development of pan-polcalcin vaccines for broad protection

These research directions build upon current methodologies and move toward more personalized and effective allergy treatments.

What methodological challenges need to be addressed in Aln G 4.0101 research?

Several methodological challenges require attention in Aln G 4.0101 research:

Addressing these challenges will require interdisciplinary approaches combining structural biology, immunology, and clinical research methodologies.