Recombinant Quercus alba Major pollen allergen Que a 1, partial

What is the molecular structure of Que a 1 and how does it compare to other tree pollen allergens?

Que a 1 is the major pollen allergen from Quercus alba (white oak) with a target protein sequence of "GVFTHESQETSVIAPARLFKALFLDSDNLIQKVLPQAIKSTEIIEGNGGP" covering amino acids 1-50 of the full protein . When expressed with an N-terminal 6His-B2M tag, the recombinant partial protein has a molecular weight of approximately 19.4 kDa .

Que a 1 shares structural homology with other tree pollen allergens, particularly those from the birch family. Based on studies of similar oak allergens, Que ac 1 from sawtooth oak (Quercus acutissima) shows 54.8% sequence identity to Bet v 1, the major birch pollen allergen . This homology reflects the evolutionary conservation of the pathogenesis-related protein family (PR-10) across different tree species, suggesting similar three-dimensional structures consisting of a seven-stranded anti-parallel β-sheet and three α-helices.

How do different expression systems affect the structure and immunoreactivity of recombinant Que a 1?

For optimal immunoreactivity, expression conditions must be carefully controlled. E. coli Rosetta-2 (DE3) strain has been successfully used with pET6xHN-N vector systems for similar oak allergens . This strain contains additional tRNAs for rare codons that may improve expression of eukaryotic proteins. Expression temperature, induction time, and IPTG concentration significantly impact protein folding and solubility.

Research indicates that recombinant Que ac 1 (from sawtooth oak) demonstrates even stronger IgE reactivity than commercial Q. alba (t7) pollen extract in some patients, suggesting that properly expressed recombinant allergens can provide enhanced diagnostic sensitivity .

What methodologies are most effective for purifying recombinant Que a 1 to ensure structural integrity?

A multi-step purification protocol typically yields the highest purity for recombinant Que a 1. Current methods achieve greater than 85% purity as determined by SDS-PAGE . The recommended approach includes:

• Immobilized metal affinity chromatography (IMAC) utilizing the N-terminal His-tag

• Size exclusion chromatography to separate monomeric protein from aggregates

• Optional endotoxin removal for immunological applications

• Buffer optimization (Tris/PBS-based buffer with 5-50% glycerol for liquid form; or with 6% Trehalose at pH 8.0 for lyophilization)

For structural validation, LC ESI MS/MS analysis confirms molecular identity of the purified protein . Circular dichroism spectroscopy can verify proper secondary structure folding compared to natural allergen extracts.

What are the optimal conditions for reconstitution and storage of recombinant Que a 1 to maintain allergenicity?

For optimal reconstitution and storage:

• Briefly centrifuge vials before opening to bring contents to the bottom

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage

• Store at -20°C/-80°C for maximum stability (liquid form is stable for up to 6 months; lyophilized powder for up to 12 months)

• Avoid repeated freeze-thaw cycles by creating working aliquots

• Working aliquots can be stored at 4°C for up to one week

These conditions maintain structural integrity and immunological activity. Research indicates that proper storage is critical for preserving epitope conformation and IgE binding capacity.

How can researchers assess the immunological equivalence between recombinant and natural Que a 1?

Assessment of immunological equivalence requires multiple complementary approaches:

• ImmunoCAP and ELISA analysis using sera from sensitized patients to compare IgE binding profiles

• Inhibition assays to determine if recombinant protein can compete with natural allergen for antibody binding

• Basophil activation tests to compare allergenic potency

• Structural analysis using circular dichroism or other spectroscopic methods

• Epitope mapping to ensure all relevant IgE-binding regions are preserved

Studies with Que ac 1 demonstrate that recombinant allergens can sometimes exhibit stronger IgE reactivity than natural pollen extracts, suggesting potential advantages for diagnostic applications . When validating a new recombinant preparation, comparison with established standards is essential for ensuring consistent experimental results.

What controls are essential when designing experiments with recombinant Que a 1?

Rigorous experimental design with recombinant Que a 1 requires:

• Positive controls: Natural Q. alba pollen extract; well-characterized recombinant allergen standards

• Negative controls: Expression system without allergen insert; buffer-only samples

• Cross-reactivity controls: Related allergens (e.g., Bet v 1) to assess specificity

• Patient sera controls: Both sensitized and non-sensitized individuals; pooled vs. individual sera

• Assay validation controls: Calibration curves using known quantities of allergen

Internal standardization is critical for comparing results across different experimental batches. LC ESI MS/MS can confirm molecular identity of the recombinant protein before experimental use .

How does Que a 1 sensitization profile compare with other oak allergens in allergic patients?

Based on studies of related oak allergens, Que a 1 likely represents the dominant sensitizing component in oak pollen allergy. Research on sawtooth oak allergens demonstrates that Que ac 1 is recognized by serum IgE in 84.0% of tree pollinosis patients, far exceeding the recognition rates of other components: Que ac 2 (12.0%), Que ac 3 (6.0%), and other minor allergens (2.0% each) .

The sensitization profile correlates with clinical manifestations:

• Allergic rhinoconjunctivitis: 83.7% of patients react to Que ac 1

• Pollen food allergy syndrome: 92.9% of patients react to Que ac 1

• Asthma: 91.0% of patients recognize Que ac 1

This data suggests that Que a 1 functions as the primary marker for oak pollen sensitization, making it particularly valuable for component-resolved diagnosis of oak pollen allergy.

What methodologies can detect cross-reactivity between Que a 1 and other allergens from related plant species?

To investigate cross-reactivity between Que a 1 and related allergens, researchers should employ:

• ImmunoCAP inhibition assays: Pre-incubation with purified allergens to measure inhibition of IgE binding

• Western blot inhibition: Visual demonstration of cross-reactive epitopes

• Basophil activation tests with cross-inhibition: Functional analysis of cross-reactivity

• Mass spectrometry epitope mapping: Identification of shared epitopes

• Computational sequence and structural analysis: Prediction of cross-reactive regions

Cross-reactivity assessment is particularly important for understanding the relationship between oak sensitization and pollen-food allergy syndrome. The significant homology between Que a 1 and Bet v 1 (54.8% for the related Que ac 1) suggests substantial cross-reactivity that may explain clinical observations of patients reacting to multiple tree pollens .

How can recombinant Que a 1 improve diagnostic sensitivity and specificity compared to whole pollen extracts?

Recombinant Que a 1 offers several advantages over whole pollen extracts:

• Enhanced diagnostic sensitivity: Studies of the related Que ac 1 show that recombinant allergen demonstrates stronger IgE reactivity than commercial Q. alba pollen extract

• Improved standardization: Consistent protein concentration and composition compared to variable natural extracts

• Elimination of confounding components: Avoids false positives from cross-reactive carbohydrate determinants (CCDs)

• Component-resolved diagnosis: Distinguishes genuine sensitization from cross-reactivity

Research indicates that Que ac 1 alone detects 84.0% of oak pollen allergic patients, with only minimal additional diagnostic value from other components . The addition of Que ac 2 increased diagnostic sensitivity by only 4.0%, confirming the primacy of the major allergen for diagnostic purposes.

What approaches can be used to create hypoallergenic variants of Que a 1 for immunotherapy development?

Development of hypoallergenic Que a 1 variants involves several methodological approaches:

• Site-directed mutagenesis of key IgE-binding residues while preserving T-cell epitopes

• Fragmentation into peptides containing T-cell epitopes but disrupting conformational B-cell epitopes

• Production of fold variants with altered secondary and tertiary structure

• Chimeric constructs combining portions of Que a 1 with non-allergenic carrier molecules

• Recombinant production of naturally occurring isoforms with reduced allergenicity

Genetically engineered recombinant allergens offer significant advantages over natural allergenic products for immunotherapy, enabling precise modification of immunological properties . These approaches aim to reduce IgE-mediated side effects while maintaining immunomodulatory potential.

How can researchers investigate the impact of environmental factors on Que a 1 expression and allergenicity?

To investigate environmental influences on Que a 1, researchers should consider:

• Comparative proteomics of pollen collected from trees growing in different environmental conditions

• Transcript analysis of Que a 1 expression under varying growth parameters

• Post-translational modification analysis under different stress conditions

• Allergenicity assessment using sera from patients from different geographical regions

• Climate chamber experiments with controlled variables (temperature, humidity, CO₂, pollutants)

Such studies could explain geographical and temporal variations in oak pollen allergy prevalence and severity. Methodology should include standardized collection protocols, rigorous environmental data recording, and consistent immunological assays to ensure reproducible results.

What novel approaches are being developed to map conformational epitopes of Que a 1?

Advanced epitope mapping techniques include:

• X-ray crystallography or cryo-electron microscopy of allergen-antibody complexes

• Hydrogen-deuterium exchange mass spectrometry to identify solvent-exposed regions

• Phage display with random peptide libraries to identify mimotopes

• Computational epitope prediction combined with experimental validation

• Single B-cell isolation and antibody cloning from allergic patients

• Alanine scanning mutagenesis with high-throughput immunoassay readouts

Precise epitope mapping is essential for understanding the molecular basis of cross-reactivity between Que a 1 and related allergens like Bet v 1, and for developing more targeted diagnostic and therapeutic approaches. The three-dimensional allergen structure determination enables rational design of hypoallergenic variants for immunotherapy .

What factors affect the stability of recombinant Que a 1 and how can they be mitigated?

Several factors impact recombinant Que a 1 stability:

• Temperature fluctuations: Store at -20°C/-80°C and avoid repeated freeze-thaw cycles

• Protein concentration: Maintain 0.1-1.0 mg/mL for optimal stability

• Buffer composition: Use Tris/PBS-based buffer with appropriate stabilizers

• Cryoprotectants: Add 5-50% glycerol for liquid storage or 6% Trehalose for lyophilization

• pH conditions: Maintain at pH 8.0 for optimal stability

• Oxidation: Include reducing agents if cysteine residues are present

For long-term stability, liquid form remains stable for up to 6 months at -20°C/-80°C, while lyophilized powder can maintain stability for up to 12 months . Working aliquots should be stored at 4°C and used within one week to maintain immunological activity.

How can researchers troubleshoot expression problems with recombinant Que a 1?

Common expression challenges and solutions include:

• Low solubility: Reduce induction temperature (16-20°C), optimize IPTG concentration, use solubility-enhancing tags

• Inclusion body formation: Employ specialized refolding protocols with controlled dilution and redox conditions

• Degradation: Add protease inhibitors, use protease-deficient host strains

• Low yield: Optimize codon usage, employ expression enhancers, test different media formulations

• Improper folding: Co-express molecular chaperones, incorporate disulfide isomerases

E. coli Rosetta-2 (DE3) strain has been successfully used for expressing similar oak allergens, suggesting it may help overcome codon bias issues . Molecular identity confirmation with LC ESI MS/MS represents an essential quality control step.

What analytical methods are most suitable for characterizing the purity and homogeneity of recombinant Que a 1 preparations?

A comprehensive analytical suite for Que a 1 characterization includes:

• SDS-PAGE: Standard method achieving >85% purity assessment

• Size exclusion HPLC: Detects aggregates and assesses homogeneity

• Mass spectrometry: Confirms molecular weight and identifies potential modifications

• Circular dichroism: Evaluates secondary structure content

• Dynamic light scattering: Measures particle size distribution

• Isoelectric focusing: Identifies charge variants

• Endotoxin testing: Essential for immunological applications

For complete characterization, a combination of these methods provides complementary information about different quality attributes. LC ESI MS/MS specifically confirms protein identity through peptide mapping , while immunological assays verify allergen functionality and epitope presentation.