Recombinant Campylobacter lari ATP synthase subunit c (atpE)
Description
Definition and Basic Characteristics
Recombinant Campylobacter lari ATP synthase subunit c (atpE) is a bioengineered protein corresponding to the native ATP synthase subunit c (UniProt ID: B9KD84) of C. lari. This protein is part of the F₀ sector of ATP synthase, critical for proton/sodium gradient-driven ATP synthesis . Key features include:
| Property | Value |
|---|---|
| Gene Name | atpE |
| Protein Length | Full-length (1–107 amino acids) |
| Molecular Weight | ~12 kDa (estimated from sequence) |
| Expression System | Escherichia coli (strain unspecified) |
| Tag | N-terminal His tag |
| Purity | >90% (SDS-PAGE validation) |
| Storage Buffer | Tris/PBS-based, 6% trehalose, pH 8.0 |
The recombinant protein includes a His tag for purification via metal affinity chromatography and is lyophilized for long-term storage .
Key Features
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His-Tagged Design: Enables efficient purification using nickel or cobalt affinity chromatography .
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Thermal Stability: While not explicitly tested for C. lari, ATP synthase subunits in related Campylobacter species show heat-induced upregulation of chaperones like dnaK and groEL, suggesting stress-responsive structural flexibility .
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Subcellular Localization: Likely embedded in the inner mitochondrial membrane or bacterial cytoplasmic membrane, though experimental validation in C. lari is limited.
Production and Purification
Recombinant atpE is typically produced in E. coli using expression vectors like pET32, which includes a thioredoxin tag for enhanced solubility and a His tag for purification . Key steps include:
Genetic Context in C. lari
Genomic analyses reveal significant diversity in C. lari isolates, including variable loci like atpA and glyA (Table 2 in source ). For example, atpA exhibits 15.5% variable sites in C. lari, indicating potential functional divergence .
| Locus | Variable Sites (%) | dN/dS Ratio | Species |
|---|---|---|---|
| atpA | 15.5 | 0.000 | C. lari |
| glyA | 17.0 | 0.035 | C. lari |
Stress Response Variability
Comparative transcriptomics show C. lari upregulates heat shock genes (dnaK, groES, groEL, clpB) differently than C. coli, suggesting divergent adaptation mechanisms to thermal stress .
Diagnostic and Therapeutic Potential
While recombinant outer membrane proteins (e.g., Omp18, MOMP) in C. jejuni are used for serological assays , atpE’s utility in diagnostics remains unexplored. Its role as a potential drug target is supported by studies in other pathogens but lacks validation in C. lari .
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will then prepare the product according to your request.
Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. For lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: cla:Cla_1201
STRING: 306263.Cla_1201
Q&A
What tags are recommended for recombinant expression of C. lari atpE?
For research applications, N-terminal His-tagged constructs have been successfully used for recombinant C. lari atpE expression . This approach enables:
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Efficient single-step purification using metal affinity chromatography
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Minimal interference with the protein's membrane-association properties
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Reliable detection using anti-His antibodies
Other tags such as GST or FLAG may be suitable depending on experimental requirements, but His-tagging remains the most validated approach for this protein.
What expression systems yield optimal results for recombinant C. lari atpE?
E. coli expression systems have proven effective for recombinant production of C. lari atpE . The following parameters are critical for optimizing expression:
| Parameter | Recommended Condition | Justification |
|---|---|---|
| Expression host | E. coli BL21(DE3) | Lacks proteases that may degrade recombinant protein |
| Induction | 0.5-1.0 mM IPTG | Balances protein yield and solubility |
| Temperature | 25-30°C post-induction | Reduces inclusion body formation |
| Growth media | Enriched media (e.g., TB or 2YT) | Supports higher cell density and protein yield |
How should researchers optimize storage conditions for recombinant C. lari atpE?
Long-term stability of recombinant C. lari atpE requires careful storage considerations:
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Store lyophilized protein at -20°C/-80°C
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For reconstituted protein, add 5-50% glycerol (final concentration) before aliquoting
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Avoid repeated freeze-thaw cycles
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Store working aliquots at 4°C for up to one week
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Use Tris/PBS-based buffer (pH 8.0) with 6% trehalose for optimal stability
For functional studies, reconstitution in lipid environments may better preserve native conformational states.
What experimental approaches effectively characterize C. lari atpE function?
Several methodologies are valuable for investigating C. lari atpE function:
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Membrane reconstitution assays: Incorporate purified atpE into liposomes to measure proton translocation activity
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Site-directed mutagenesis: Identify critical residues for function by introducing targeted mutations
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Protein-protein interaction studies: Examine assembly with other ATP synthase subunits
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Cross-linking experiments: Determine oligomeric states and structural arrangements
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Comparative expression analysis: Evaluate expression under various environmental conditions using RNA-seq
These approaches provide complementary insights into both structural and functional properties of the protein.
How does heat stress affect C. lari atpE expression and function?
Transcriptomic analysis of C. lari under heat stress (46°C) reveals complex expression patterns affecting multiple cellular processes. While atpE itself wasn't specifically highlighted among differentially expressed genes in available studies, related ATP synthase components show altered expression during heat stress .
The heat stress response in C. lari involves:
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Upregulation of chaperones (dnaK, groES, groEL, clpB) that may assist in proper folding of membrane proteins including atpE
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Altered expression of genes involved in cell wall/membrane/envelope biogenesis
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Changes in energy metabolism pathways that likely affect ATP synthase function
These changes suggest that functional ATP synthase complexes, including atpE, may be maintained during stress through compensatory mechanisms rather than direct upregulation.
How does the transcriptional response of atpE differ between Campylobacter species under stress conditions?
Transcriptomic studies comparing C. lari and C. coli under heat stress (46°C) demonstrate substantial differences in gene expression patterns. Approximately 20% of genes show differential expression in both species, but with distinct profiles .
While specific atpE expression data is limited, the broader patterns suggest:
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C. lari exhibits unique regulatory mechanisms compared to other Campylobacter species
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Heat stress response appears to be species-specific rather than conserved across the genus
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ATP synthesis and energy metabolism show divergent regulation patterns between species
These differences highlight the importance of species-specific investigations rather than extrapolating findings across Campylobacter species.
How can C. lari atpE be utilized in antimicrobial resistance studies?
The ATP synthase complex represents a potential target for antimicrobial development. For C. lari specifically:
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The membrane-embedded nature of atpE makes it a candidate for membrane-targeting antimicrobials
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Species-specific sequence variations could potentially be exploited for selective targeting
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Expression changes during stress conditions might reveal vulnerabilities for therapeutic intervention
Research approaches include:
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Screening compound libraries for specific inhibition of C. lari atpE
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Investigating synergistic effects with other antimicrobials
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Examining potential interactions with efflux pump systems like cmeA and cmeB that contribute to antimicrobial resistance in Campylobacter species
What methodological challenges exist when investigating protein-protein interactions involving C. lari atpE?
The investigation of atpE interactions presents several technical challenges:
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Membrane localization: The hydrophobic nature of atpE complicates traditional protein-protein interaction assays
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Complex assembly: ATP synthase involves multiple subunits assembled in a specific stoichiometry
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Detergent sensitivity: Extraction conditions can disrupt native interactions
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Expression levels: Natural abundance may be insufficient for detection by standard methods
Recommended approaches include:
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Chemical cross-linking followed by mass spectrometry
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Bacterial two-hybrid systems adapted for membrane proteins
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Proximity-based labeling methods (BioID, APEX)
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Native membrane extraction using mild detergents followed by blue native PAGE
How might structural biology approaches advance our understanding of C. lari atpE?
While no high-resolution structure specific to C. lari atpE is currently available, several structural biology approaches could provide valuable insights:
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Cryo-electron microscopy: Could reveal the arrangement of atpE within the complete ATP synthase complex
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Solid-state NMR: Particularly suited for membrane proteins like atpE
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Molecular dynamics simulations: Can predict species-specific structural features based on the known sequence
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Hydrogen-deuterium exchange mass spectrometry: May reveal dynamic regions important for function
These approaches would address the current knowledge gap regarding specific structural features of C. lari atpE compared to better-characterized bacterial ATP synthases.
What is the relationship between C. lari atpE expression and pathogenicity?
The connection between ATP synthase function and pathogenicity in Campylobacter remains an open research question. Future investigations might explore:
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The role of atpE in adaptation to host environments
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Potential correlation between atpE expression and virulence factor production
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Survival advantages conferred by ATP synthase regulation during infection
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Comparative analysis of atpE sequence variants across clinical and environmental isolates
Given the essential role of ATP synthesis in bacterial metabolism, understanding how C. lari regulates this process during infection could reveal new insights into pathogenesis mechanisms.
