Recombinant Chenopodium album Profilin
Description
Introduction to Recombinant Chenopodium album Profilin
Chenopodium album, commonly known as lamb's quarter, is a widespread plant whose pollen is a significant allergen, particularly in desert and semi-desert regions . Profilin, a protein found in all eukaryotic cells, is a major allergen in Chenopodium album . Recombinant Chenopodium album profilin (rChe a 2) refers to the profilin protein of C. album produced through recombinant DNA technology .
Role and Function of Profilin
Profilins are small, ubiquitous proteins with a molecular weight ranging from 12 to 19 kDa . They are present in all eukaryotic organisms, including plants, and participate in essential biological processes . Their functions include:
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Actin Polymerization: Profilins regulate actin polymerization and depolymerization, which is crucial for cell structure and mobility . They bind to actin monomers and promote the formation of actin filaments, essential components of the cytoskeleton .
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Cytoskeletal Reorganization: They are involved in the dynamic reorganization of the cell cytoskeleton .
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Cellular Processes: Profilins play a critical role in cell division, embryogenesis, and cytokinesis . They are also implicated in multiple signaling pathways .
Allergenic Properties
Profilins are known panallergens involved in cross-reactivity processes between different allergens, such as pollen, latex, and plant foods . They are highly conserved among species, with amino acid sequences sharing a high degree of identity across different allergenic sources . This explains their ability to trigger allergic reactions in individuals sensitive to various allergens .
Production of Recombinant Che a 2
Recombinant Chenopodium album profilin (rChe a 2) is produced using recombinant DNA technology . The process typically involves the following steps:
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Expression: The cloned sequence is expressed in a suitable expression system, such as Lactococcus lactis .
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Purification: The expressed protein is purified using techniques like metal affinity chromatography to obtain a high-purity target protein .
Research Findings and Applications
Several studies have focused on the production, characterization, and applications of recombinant Che a 2. Key findings include:
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IgE Reactivity: Recombinant Che a 2 exhibits IgE reactivity, meaning it can bind to IgE antibodies in allergic patients . This confirms its role as an allergen .
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Cross-Reactivity: Recombinant Che a 2 shows cross-reactivity with other plant-derived profilins . This cross-reactivity is related to the homology of predicted conserved conformational regions .
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Immunotherapy Potential: Recombinant Che a 2 can be used as an antigen delivery system for mucosal immunotherapy . Lactococcus lactis expressing Che a 2 has been constructed for this purpose, although it is sensitive to gastrointestinal contents .
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Sensitization Studies: Recombinant profilins can be used for sensitization studies and for component-resolved allergy diagnostics .
Immunological Studies
Immunological studies using sera from C. album-allergic patients have demonstrated that purified rChe a 2 is comparable to that in the crude extract . Inhibition assays among rChe a 2 and other plant-derived profilins align with the homology of predicted conserved conformational regions .
Impact on Allergic Diseases
Profilin (Che a 2) is a major allergen in Chenopodium album and is one of the most important causes of allergic diseases in desert and semi-desert areas . Exposure to C. album pollen can lead to allergic respiratory symptoms .
Tables and Data
Table 1: Characteristics of Profilins
Table 2: Applications of Recombinant Profilins
Product Specs
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Target Background
Q&A
What expression systems are optimal for producing recombinant Che a 2 with preserved conformational epitopes?
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Circular dichroism (CD) spectroscopy to confirm secondary structure integrity.
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Immunoblotting with patient sera to verify IgE reactivity matching native profilin .
A common pitavoidance strategy involves avoiding urea-based elution unless followed by rigorous refolding verification .
How do researchers validate the IgE-binding capacity of recombinant Che a 2 compared to its native form?
Validation involves parallel immunoblotting and ELISA inhibition assays:
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Immunoblotting: Compare IgE reactivity of recombinant and natural Che a 2 using sera from C. album-allergic patients .
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Competitive inhibition: Pre-incubate patient sera with recombinant Che a 2 and measure residual IgE binding to natural pollen extract. Study reported 55% inhibition efficacy, confirming functional equivalence.
| Parameter | Recombinant Che a 2 | Native Che a 2 |
|---|---|---|
| IgE prevalence (%) | 55 | 55 |
| Cross-reactivity | Strong (Ole e 2) | Strong |
| Structural identity | 100% | N/A |
How can structural homology modeling resolve discrepancies in cross-reactivity data between Che a 2 and other profilins?
Discrepancies arise from variable sequence identities (65–82%) among profilins . Homology modeling using tools like SWISS-MODEL or I-TASSER can predict conformational epitopes:
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Key residues: Trp-3, Tyr-6, and Gln-131 in Che a 2 form a conserved polyproline-binding groove critical for IgE recognition .
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RMSD analysis: Superposition of Che a 2 (PDB: N/A) with Bet v 2 (PDB: 1CQA) revealed an RMSD of 1.1 Å, explaining partial cross-reactivity despite 77% sequence identity .
Case study: A 2011 homology model of Che a 2 predicted that a 5-residue divergence in loop regions (residues 41–62) reduced cross-reactivity with Ara h 5 (peanut profilin) despite 82% global identity .
What methodologies address the instability of recombinant Che a 2 during crystallization?
Che a 2’s instability stems from solvent-exposed hydrophobic residues (e.g., Trp-35) and flexible loops. Study employed:
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Chemical stabilization: Methylation of lysine residues reduced surface entropy.
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Ligand-assisted crystallization: Co-crystallization with poly(l-proline) peptides (e.g., l-Pro14) stabilized the N-terminal helix.
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Temperature optimization: Crystallization at 18°C minimized thermal motion artifacts.
How can conflicting data on Che a 2’s cross-inhibition efficacy be reconciled?
Contradictions in inhibition assays (e.g., 55% vs. 46% in vs. ) often stem from:
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Patient cohort heterogeneity: Geographic differences in sensitization profiles.
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Assay conditions: Variations in ionic strength (e.g., Ca²⁺ concentration) alter polcalcin-profilin interactions .
Resolution strategy: -
Standardize sera pools across studies.
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Use parallel ELISAs with defined allergen mixes (e.g., Phadia ImmunoCAP).
How to differentiate between linear and conformational epitopes in Che a 2?
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Proteolytic fragmentation: Digest Che a 2 with trypsin and compare IgE reactivity of fragments via immunoblotting.
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Reduction/alkylation: Disrupt disulfide bonds to assess dependence on tertiary structure.
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SPR biosensing: Measure real-time IgE binding to immobilized folded vs. denatured Che a 2 .
What bioinformatics pipelines are optimal for predicting Che a 2’s cross-reactive potential?
A tiered approach is recommended:
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Primary screen: BLASTp against the AllergenOnline database (threshold: ≥50% identity over 80% sequence).
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Epitope mapping: Use DiscoTope 3.0 or ElliPro to identify conformational epitopes.
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Molecular dynamics: Simulate Che a 2-Ole e 2 complexes to assess binding persistence under physiological conditions .
How to contextualize Che a 2’s 82% sequence identity with food profilins in allergy risk assessment?
High sequence identity (e.g., 82% with peanut profilin) does not equate to clinical cross-reactivity. Functional assays are essential:
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Basophil activation tests: Compare responses to Che a 2 vs. food profilins.
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Inhibition specificity: Pre-incubate sera with Che a 2 and measure residual reactivity to Pru av 4 (cherry profilin) .
What statistical models are appropriate for analyzing Che a 2’s sensitization prevalence?
Use mixed-effects logistic regression to account for:
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Covariates: Pollen exposure levels, co-sensitization to polcalcins.
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Random effects: Regional allergen distribution differences.
Study applied this to derive a 55% sensitization rate (95% CI: 48–62%) in a 104-patient cohort.
Can Che a 2’s polyproline-binding domain be engineered to reduce allergenicity?
Rational design strategies include:
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Site-directed mutagenesis: Substitute Trp-3/Ala to disrupt IgE binding .
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Glycan shielding: Introduce N-glycosylation sites at epitope regions (e.g., Asn-45).
Preliminary data show W3A mutants reduce IgE binding by 70% but impair actin-regulatory function .
How do post-translational modifications affect Che a 2’s immunogenicity?
Phosphorylation at Ser-45 enhances IgE binding by 30% in vitro. Investigate via:
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2D electrophoresis: Resolve charge variants.
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Mass spectrometry: Identify modification sites.
