Pen a 1.0101
Description
Biochemical Characteristics
Pen a 1.0101 belongs to the tropomyosin family, a group of actin-binding proteins essential for muscle contraction regulation. Key attributes include:
| Property | Details | Source |
|---|---|---|
| Molecular Weight | 34–36 kDa | |
| Sequence Length | 284 amino acids | |
| Protein Family | Tropomyosin | |
| GenBank Nucleotide ID | DQ151457 | |
| UniProt Accession | Q3Y8M6 |
The protein’s structure includes coiled-coil domains characteristic of tropomyosin, enabling its interaction with actin filaments. Recombinant forms of Pen a 1.0101 have been expressed in heterologous systems to study its allergenicity and functional properties .
Allergenicity and Clinical Relevance
Pen a 1.0101 is a high-potency allergen linked to severe IgE-mediated hypersensitivity reactions:
The allergen’s stability under heat and digestion contributes to its persistence in cooked shrimp, exacerbating cross-reactivity in pan-crustacean allergies .
Research Applications
Pen a 1.0101 is widely used in immunological and biochemical studies:
Commercial preparations of Pen a 1.0101 (e.g., catalog B2013762) are available as purified antigens for immunological assays .
Key Research Findings
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Structural and Functional Properties
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Allergenicity Mechanisms
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Diagnostic and Therapeutic Implications
References
Product Specs
Penaeus aztecus, or brown shrimp, contains Tropomyosin Pen a 1.0101, its only known major allergen. This panallergen is a highly conserved and soluble muscle protein characterized by an alpha-helical homodimeric, coiled-coil structure.
Recombinant Tropomyosin Pen a 1.0101, expressed in SF9 cells, is a glycosylated polypeptide chain with a calculated molecular mass of 38kDa. This protein is expressed with a 10xHis tag and purified using proprietary chromatographic techniques.
Pen a 1.0101 is supplied in a 20mM HEPES buffer with a pH of 7.9, 200mM NaCl, and 6M Urea.
For optimal storage, keep at 4°C if the entire vial will be used within 2-4 weeks. For longer storage periods, store frozen at -20°C. Avoid repeated freeze-thaw cycles.
Purity is determined to be greater than 95% as assessed by SDS-PAGE.
Sf9 insect cells.
Q&A
What is Pen a 1.0101 and what are its fundamental biochemical properties?
Pen a 1.0101 is a recombinant allergen protein from Penaeus aztecus (brown shrimp) primarily used for in vitro research and manufacturing applications. It belongs to the tropomyosin family of proteins and is characterized by its ability to bind IgE-type human antibodies, making it an important molecule in allergy research .
The key biochemical properties of Pen a 1.0101 include:
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Molecular weight: 34 kDa
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Isoelectric point: pH 5.02
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Protein purity: >80% as determined by SDS-PAGE
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Functional activity: Verified through Western blot analysis with patient samples and immunodot analyses with positive/negative control samples
For research applications, Pen a 1.0101 is typically expressed as a recombinant protein with a deca-histidine purification tag, facilitating efficient isolation and functional studies while maintaining its immunological characteristics.
How is Pen a 1.0101 expressed and purified for research purposes?
The expression and purification of Pen a 1.0101 follows a specific methodological approach:
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Expression system: Pen a 1.0101 is expressed using recombinant baculovirus (Autographa californica multiple nuclear polyhedrosis virus; AcMNPV) infection of Spodoptera frugiperda Sf9 insect cells .
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Expression construct: The process utilizes full-length cDNA coding for Pen a 1.0101 fused to a deca-histidine purification tag, which facilitates subsequent purification steps .
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Purification methodology: While not explicitly detailed in the search results, the presence of a deca-histidine tag suggests immobilized metal affinity chromatography (IMAC) is likely employed for purification.
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Quality control: The purified protein undergoes multiple quality control procedures:
This methodological approach ensures consistent production of high-quality Pen a 1.0101 suitable for sensitive immunological research applications.
What are the optimal storage and handling conditions for Pen a 1.0101?
For maintaining the stability and functionality of Pen a 1.0101 in research settings, specific storage and handling protocols should be followed:
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Buffer composition: The recommended storage buffer should maintain a neutral to slightly alkaline pH and contain 6 M urea to ensure protein stability .
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Storage temperature: The optimal storage temperature is -70°C or below .
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Handling precautions: Repeated freeze/thaw cycles should be avoided as they can compromise protein integrity and function .
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Thawing procedure: While not explicitly mentioned in the search results, standard protein handling protocols suggest thawing frozen protein samples on ice immediately before use.
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Working solution preparation: Dilutions should be prepared fresh in appropriate buffers depending on the specific application (e.g., PBS with low detergent concentrations for immunoassays).
Adherence to these storage and handling guidelines is crucial for maintaining the structural integrity and functional properties of Pen a 1.0101 during experimental procedures.
How does the gene structure of Pen a 1.0101 compare to tropomyosin allergens from other crustacean species?
Comparative genomic analysis reveals significant structural similarities and evolutionary relationships among tropomyosin genes across decapod crustaceans:
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Conserved gene architecture: The gene structure of Pen a 1.0101 (from Penaeus aztecus) shares remarkable similarity with Pen m 1.0101 (from Penaeus monodon), Pro c 1.0101 (from Procambarus clarkii), and Hom a 1.0101/Hom a 1.0102 (from Homarus americanus) .
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Exon organization: All these tropomyosin genes feature a multi-exon structure with a consistent pattern of exon combinations. The genes contain numbered exons (1-9), with some exons having alternative forms (denoted by letters, e.g., 2a, 4b) .
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Single gene presence: A notable finding is that all examined species possess only a single tropomyosin gene within their entire genomes, regardless of genome size. This suggests that functional diversity arises not from gene duplication but through increased gene complexity and isoform generation .
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Evolutionary implications: The comparative analysis indicates that tropomyosin diversification through alternative splicing likely emerged later in crustacean evolution and primarily exists within decapods .
The gene structure comparison provides crucial insights into the evolutionary mechanisms that have shaped allergen diversity in crustaceans, with direct implications for understanding cross-reactivity patterns in shellfish allergies.
What role does alternative splicing play in tropomyosin diversity among decapods?
Alternative splicing represents a critical mechanism for generating functional diversity in tropomyosin proteins among decapod crustaceans:
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Isoform generation: Rather than gene duplication, decapods achieve tropomyosin subfunctionalization through increased gene complexity and the generation of multiple isoforms via alternative splicing .
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Exon utilization patterns: The research identifies specific exons with alternative forms, including exons 2, 4, 5, 7, and 9 in species like P. clarkii. These alternative exons demonstrate varying transcription levels, as illustrated by the coverage analysis (Figure 1C in search result ) .
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Differential exon expression: Transcriptome mapping reveals that certain alternative exons show markedly different expression levels. For example, in P. clarkii, exon 2 alternatives show 100% coverage while exon 9 alternatives show only 37% coverage, indicating differential usage in mRNA transcripts .
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Evolutionary significance: The findings suggest that alternative splicing as a mechanism for tropomyosin diversification emerged relatively late in crustacean evolution, primarily within the decapod order .
This sophisticated alternative splicing machinery provides decapods with the ability to generate multiple protein isoforms from a single gene, potentially contributing to tissue-specific functions and allergenic properties of tropomyosin proteins.
How can researchers experimentally validate the IgE binding epitopes in Pen a 1.0101?
The experimental validation of IgE binding epitopes in Pen a 1.0101 requires a systematic approach combining multiple methodologies:
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Peptide microarray analysis: Researchers can synthesize overlapping peptides spanning the entire Pen a 1.0101 sequence and test them against serum samples from shellfish-allergic patients to identify linear IgE-binding epitopes.
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Site-directed mutagenesis: Key amino acid residues within predicted epitopes can be mutated, followed by immunological testing to determine their contribution to IgE binding. This approach is particularly useful for confirming the functional significance of specific residues.
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Cross-reactivity studies: Comparing IgE binding patterns between Pen a 1.0101 and homologous tropomyosins from other species (e.g., Pen m 1.0101, Pro c 1.0101) can help identify conserved epitopes responsible for cross-reactivity. The expressed recombinant protein can be utilized for these immunodot analyses with positive/negative patient samples .
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Recombinant allergen fragments: Creating and testing recombinant fragments of Pen a 1.0101 can help map the distribution of conformational and linear epitopes throughout the protein structure.
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Structural biology approaches: Three-dimensional structural analysis using X-ray crystallography or NMR spectroscopy, combined with in silico epitope prediction and molecular docking, can further enhance understanding of conformational epitopes.
These methodological approaches provide complementary data that together create a comprehensive map of IgE binding epitopes on Pen a 1.0101, valuable for developing improved diagnostics and potential immunotherapies for shellfish allergy.
What techniques can be employed to study the structural changes in Pen a 1.0101 under different environmental conditions?
Understanding how environmental factors affect the structural integrity and allergenicity of Pen a 1.0101 requires sophisticated biophysical and biochemical approaches:
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Circular Dichroism (CD) Spectroscopy: This technique allows researchers to monitor changes in the secondary structure (α-helices, β-sheets) of Pen a 1.0101 under varying conditions such as pH, temperature, denaturants, or food processing methods.
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Differential Scanning Calorimetry (DSC): DSC can determine the thermal stability and unfolding patterns of Pen a 1.0101, providing insights into how heat treatments affect its allergenicity.
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Fourier Transform Infrared Spectroscopy (FTIR): This method provides detailed information about protein secondary structure and can track structural changes induced by environmental modifications.
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Limited Proteolysis coupled with Mass Spectrometry: This approach can identify regions of the protein that become more or less susceptible to enzymatic digestion under different conditions, indicating conformational changes.
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IgE-binding assays following treatments: Immunological testing of Pen a 1.0101 after exposure to different conditions can correlate structural changes with alterations in allergenic potential.
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Nuclear Magnetic Resonance (NMR) Spectroscopy: For more detailed structural analysis, NMR can provide atomic-level information about structural perturbations under various conditions.
When designing such experiments, researchers should consider that Pen a 1.0101 is optimally stored in buffer with neutral to slightly alkaline pH containing 6 M urea , suggesting that the protein may have specific stability requirements that should be accounted for in experimental designs.
What comparative analyses can be performed between Pen a 1.0101 and other tropomyosin isoforms?
Comprehensive comparative analyses between Pen a 1.0101 and other tropomyosin isoforms can reveal important evolutionary relationships and functional differences:
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Sequence alignment and phylogenetic analysis: Comparing the amino acid sequences of Pen a 1.0101 with homologs like Pen m 1.0101, Pro c 1.0101, and Hom a 1.0101/1.0102 can establish evolutionary relationships and identify conserved regions potentially important for allergenicity .
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Exon usage comparison: Analysis of exon combinations between different tropomyosin isoforms, as illustrated for Pen m 1.0101, Pro c 1.0101, and Hom a 1.0101/1.0102, can reveal species-specific patterns of alternative splicing .
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Cross-species immunological reactivity: Comparative immunological studies can determine the degree of cross-reactivity between Pen a 1.0101 and tropomyosins from other species, which has direct clinical relevance for allergic individuals.
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Functional domain analysis: Mapping functional domains across different tropomyosin isoforms can identify conserved regions responsible for specific biological functions versus regions that may have undergone adaptive evolution.
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Structural comparison: Three-dimensional structural comparisons can reveal subtle differences in protein folding that may influence allergenicity despite high sequence similarity.
The table below summarizes key comparative features among tropomyosin isoforms from different decapod species:
| Feature | Pen a 1.0101 | Pen m 1.0101 | Pro c 1.0101 | Hom a 1.0101 | Hom a 1.0102 |
|---|---|---|---|---|---|
| Source Species | Penaeus aztecus | Penaeus monodon | Procambarus clarkii | Homarus americanus | Homarus americanus |
| Common Name | Brown shrimp | Black tiger shrimp | Red swamp crayfish | American lobster | American lobster |
| Gene Structure | Single gene with alternative exons | Single gene with alternative exons | Single gene with alternative exons | Single gene with alternative exons | Single gene with alternative exons |
| Alternative Splicing | Present | Present | Present | Present | Present |
| Molecular Weight | 34 kDa | Similar to Pen a 1.0101 | Similar to Pen a 1.0101 | Similar to Pen a 1.0101 | Similar to Pen a 1.0101 |
These comparative analyses provide crucial insights into the molecular evolution of tropomyosin allergens and help explain patterns of clinical cross-reactivity among different shellfish species.
Product Science Overview
Tropomyosin Pen a 1.0101 is a recombinant protein derived from the brown shrimp, Penaeus aztecus. It is a significant allergen and is known for its highly conserved and soluble muscle protein properties. This protein is of particular interest due to its role as a major allergen in shrimp, making it a critical subject of study for allergy research and diagnostics.
Tropomyosin Pen a 1.0101 is characterized by its alpha-helical homodimeric, coiled-coil structure. This structural configuration is essential for its function and stability. The recombinant form of this protein is produced in Sf9 insect cells and is glycosylated, resulting in a polypeptide chain with a calculated molecular mass of approximately 38 kDa .
The protein is expressed with a 10xHis tag, which facilitates its purification through proprietary chromatographic techniques. The final product is a sterile, filtered, colorless solution supplied in a buffer containing 20 mM HEPES, pH 7.9, 200 mM sodium chloride, and 6 M urea .
The recombinant Tropomyosin Pen a 1.0101 is produced using Sf9 insect cells, a common expression system for producing recombinant proteins. This system allows for proper folding and post-translational modifications, such as glycosylation, which are crucial for the protein’s functionality and stability.
The purification process involves multiple chromatographic steps to ensure a high degree of purity. The final product has a purity greater than 95%, as determined by SDS-PAGE .
Tropomyosin Pen a 1.0101 is primarily used in allergy research. As the major allergen in brown shrimp, it is a critical component in the development of diagnostic tests and therapeutic approaches for shrimp allergies. The recombinant form allows for consistent and reliable results in laboratory settings.
