Recombinant Brucella suis ATP synthase subunit delta (atpH)
Description
Research Applications and Experimental Insights
Recombinant atpH is utilized in structural and functional studies of ATP synthase. Key findings include:
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Interaction Studies: Co-expression with subunits α and β in heterologous systems enables reconstitution of functional F₁ sectors for biochemical assays.
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Antigenic Potential: While not directly studied, ATP synthase subunits are recognized as vaccine targets in other pathogens, though Brucella LPS remains a primary focus for immunization strategies .
Table 2: Comparative Analysis of ATP Synthase Subunits in Brucella suis
Challenges and Gaps in Research
Despite its conserved role, Brucella suis atpH remains understudied compared to other ATP synthase subunits. Key gaps include:
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In Vivo Function: No studies directly link atpH to Brucella pathogenesis or intracellular survival.
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Structural Data: High-resolution crystal structures for Brucella ATP synthase are absent, limiting mechanistic insights.
Product Specs
Target Background
KEGG: bmt:BSUIS_B1278
Q&A
Experimental Design for Studying Recombinant Brucella suis ATP Synthase Subunit Delta (atpH)
Q: How should I design experiments to study the role of recombinant Brucella suis ATP synthase subunit delta (atpH) in bacterial pathogenesis?
A:
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Objective: Investigate how atpH affects bacterial survival and virulence within host cells.
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Methods:
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Expression and Purification: Clone the atpH gene into an expression vector (e.g., pET28a) and express it in E. coli. Purify the protein using affinity chromatography.
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Cell Culture Assays: Use macrophage or epithelial cell lines to study intracellular survival and replication of Brucella suis expressing recombinant atpH.
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Immunofluorescence Microscopy: Visualize the localization of atpH within host cells.
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Western Blotting: Confirm protein expression levels.
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Data Analysis: Compare survival rates and protein expression levels between wild-type and atpH-expressing strains.
Data Contradiction Analysis in Recombinant Protein Studies
Q: How can I address contradictory data regarding the role of atpH in Brucella pathogenesis?
A:
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Review Literature: Compare methodologies and results from different studies.
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Experimental Replication: Repeat experiments under controlled conditions to verify findings.
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Statistical Analysis: Use statistical tools to assess significance and variability in data.
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Considerations: Differences in host cell types, bacterial strains, or expression systems might contribute to discrepancies.
Advanced Research Questions on atpH Function
Q: What advanced research questions can be explored regarding the function of atpH in Brucella suis?
A:
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Protein-Protein Interactions: Investigate interactions between atpH and other bacterial proteins or host factors using co-immunoprecipitation or yeast two-hybrid assays.
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Structural Biology: Determine the crystal structure of atpH to understand its mechanism of action.
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Gene Knockout Studies: Create atpH knockout mutants to assess its role in bacterial survival and virulence.
Methodological Considerations for Recombinant Protein Expression
Q: What methodological considerations are crucial for expressing recombinant Brucella suis atpH in E. coli?
A:
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Vector Selection: Choose vectors like pET28a that allow for high-level expression and easy purification.
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Optimization of Expression Conditions: Test different temperatures, IPTG concentrations, and induction times to optimize protein yield.
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Purification Techniques: Use affinity chromatography followed by size exclusion chromatography for high purity.
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Protein Stability: Assess stability under various storage conditions to ensure long-term usability.
Data Interpretation and Validation
Q: How can I validate and interpret data from experiments involving recombinant atpH?
A:
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Validation Techniques: Use Western blotting and mass spectrometry to confirm protein identity and purity.
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Biological Replicates: Perform experiments with multiple biological replicates to ensure reproducibility.
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Statistical Analysis: Apply appropriate statistical tests to assess significance of findings.
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Literature Comparison: Compare results with existing literature to contextualize findings.
Integration with Other Brucella Pathogenesis Studies
Q: How can studies on atpH be integrated with broader research on Brucella pathogenesis?
A:
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Pathway Analysis: Investigate how atpH interacts with other known virulence factors in Brucella.
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Comparative Genomics: Compare atpH sequences across different Brucella species to identify conserved regions.
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Vaccine Development: Explore whether atpH could serve as a target for vaccine development by assessing its immunogenicity.
Challenges in Studying Recombinant atpH
Q: What challenges might researchers face when studying recombinant Brucella suis atpH, and how can they be addressed?
A:
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Protein Instability: Use stabilizing agents like glycerol and store at low temperatures to maintain protein integrity.
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Low Yield: Optimize expression conditions and consider using different expression systems.
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Contamination Risks: Implement strict biosafety protocols when handling Brucella-derived materials.
Future Directions for atpH Research
Q: What are potential future directions for research on recombinant Brucella suis atpH?
A:
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Structural Studies: Investigate the structural dynamics of atpH to understand its function.
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Host-Pathogen Interactions: Study how atpH influences host cell signaling pathways.
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Therapeutic Targets: Explore whether atpH could be targeted for therapeutic interventions against brucellosis.
Collaborative Research Opportunities
Q: How can researchers collaborate effectively on studies involving recombinant atpH?
A:
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Interdisciplinary Teams: Form teams with microbiologists, biochemists, and structural biologists.
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Shared Resources: Share expression systems, purification protocols, and analytical tools.
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Joint Publications: Collaborate on publications to integrate diverse expertise and findings.
Ethical Considerations in atpH Research
Q: What ethical considerations should researchers be aware of when conducting studies on recombinant Brucella suis atpH?
A:
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Biosafety: Ensure strict adherence to biosafety guidelines when handling Brucella-derived materials.
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Animal Welfare: Follow ethical guidelines for animal studies, ensuring minimal distress and adherence to regulatory standards.
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Data Sharing: Share data responsibly, considering implications for public health and research integrity.
